Systems and methods for genetic and biological analysis

ABSTRACT

The invention relate to systems and methods for sequencing polynucleotides, as well as detecting reactions and binding events involving other biological molecules. The systems and methods may employ chamber-free devices and nanosensors to detect or characterize such reactions in high-throughput. Because the system in many embodiments is reusable, the system can be subject to more sophisticated and improved engineering, as compared to single use devices.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalApplication No. 61/491,081 filed May 27, 2011, U.S. ProvisionalApplication No. 61/565,651 filed Dec. 1, 2011, U.S. ProvisionalApplication No. 61/620,381 filed Apr. 4, 2012, and U.S. application Ser.No. 13/397,581 filed Feb. 15, 2012, each of which is hereby incorporatedby reference in its entirety. This PCT Application is being filed withthe U.S. Receiving Office on May 29, 2012, since Monday May 28^(th) wasa federal holiday.

This Application is further related to PCT/US2011/054769, which ishereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

Aspects of this invention were made with government support under aQualifying Therapeutic Discovery Grant awarded by the IRS, inconjunction with the Department of Health and Human Services. The U.S.government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

Methods for quick and cost effective genetic and biological analysis,including high-throughput DNA sequencing, remain an important aspect ofadvancing personalized medicine and diagnostic testing. Current highthroughout or miniaturized systems have limitations. For example,current systems for DNA sequencing, including those that employ opticaldetection, are cumbersome and expensive, and have limited throughput.While some systems use sensors and sequencing flow cells to addressthese limitations, these are generally one-time use disposables, whichsubstantially increases the cost to the user and limits the complexityof the sensor, since the sensor must be cost effectively manufacturedfor a single use. Emulsion PCR provides some advantages, howeversequencing clonal DNA populations can exhibit limited accuracy whensequencing does not proceed “in phase” throughout the clonal population,which in-turn can lead to, in effect, short read lengths.

A need exists for systems and methods for genetic and biologicalanalysis, and in particular, methods and systems for highly parallel orclonal sequencing reactions that are both sensitive and cost effective.

BRIEF SUMMARY OF THE INVENTION

The aspects and embodiments described herein relate to systems andmethods for sequencing polynucleotides, as well as detecting reactionsand binding events involving other biological molecules. The systems andmethods may employ chamber-free devices and/or nanosensors to detectand/or characterize such reactions in high-throughput. Because thesystem in many embodiments is reusable, the system can be subject tomore sophisticated and improved engineering, as compared to single usedevices.

In some embodiments, the invention provides methods and systems forsequencing polynucleotides, which may be individual double or singlestranded polynucleotides, or in other embodiments are clonal populationsof polynucleotides. For example, one aspect of the invention provides amethod for parallel or clonal polynucleotide sequencing, the methodcomprising: sequencing a first portion of a population of targetpolynucleotides, correcting for phase error, and then sequencing asecond downstream portion of the population of target polynucleotides.In various embodiments, the polynucleotide sequencing may involve one ormore of: clonal sequencing of a bead array, electronic detection ofnucleotide incorporation, and an electronic well to isolate orconcentrate reaction components.

For example, phase error may be corrected by adding a combination ofthree nucleotide bases to halt the population of polynucleotides at thefirst occurrence of the excluded base. Phase error may also be correctedthrough the combination and/or order of incorporation reactions asdescribed in detail herein. Alternatively or in addition, phase errormay be corrected by reversibly incorporating, into the in-phasepolynucleotide strands, a chain terminating nucleotide. Alternatively orin addition, phase error may be corrected by adding one or moreoligonucleotide clamps, the clamp(s) hybridizing to the targetpolynucleotides to halt the sequencing reaction. In some embodiments,the clamp is denatured, destabilized, or degraded to continue thesequencing reaction. In other embodiments, at least one clamp has a 3′terminating nucleotide that cannot be extended, and thus upon removal ofthe 3′ terminating nucleotide, the clamp becomes a primer for subsequentdownstream sequencing.

Re-phasing can occur at regular intervals, or alternatively, thereaction can be monitored for loss of signal, and rephasing conducted torestore sequencing signal.

In another aspect, the invention provides a method for reducing leadingphase error in parallel or clonal polynucleotide sequencing. The methodaccording to this aspect comprises sequencing a population of targetpolynucleotides in the presence of a competitive reaction, where thecompetitive reaction comprises nucleotide bases or nucleotidederivatives for all four nucleotide bases. Generally, three of the fournucleotide bases will be unincorporable into the growing polynucleotidestrand, thereby decreasing the propensity of the polymerase toincorporate incorrect nucleotides. According to this aspect, thepolynucleotide sequencing may optionally involve one or more of: clonalsequencing of a bead array, electronic detection of nucleotideincorporation, and an electronic well to isolate or concentrate reactioncomponents. Various nucleotide derivatives are known and describedherein which may be bound by the polymerase, but not incorporated intothe growing polynucleotide strand.

In still other aspects, the invention provides a method for reducinglagging phase error in parallel or clonal polynucleotide sequencingreactions. In accordance with this aspect, the method comprisesstockpiling polymerase enzyme on or near a population of targetpolynucleotides during a sequencing reaction, such that polymerase issubstantially available for each active polymerization site.Alternatively or in addition, the method comprises binding a repairprotein or single stranded DNA binding protein to the population oftarget polynucleotides. Optionally, the polynucleotide sequencingreaction involves one or more of: clonal sequencing of a bead array,electronic detection of nucleotide incorporation, and an electronic wellto isolate or concentrate reaction components. The stockpiling can be aresult of the native binding of the polymerase to primers, includingnon-extendable primers hybridized to the template polynucleotides.

In still other aspects, the invention provides methods for repeatednucleotide sequencing, such that several sequencing runs can be analyzedfor sequence data. According to this aspect, the method comprisesproviding a circularized DNA sequencing template, and sequencing thetemplate by determining the sequence of incorporation of nucleotides bya DNA polymerase having 5′ to 3′ exonuclease activity. This aspect mayalso optionally involve one or more of: clonal sequencing of a beadarray, electronic detection of nucleotide incorporation, and anelectronic well to isolate or concentrate reaction components. The DNApolymerase according to this aspect may be highly processive and havereduced exonuclease activity. The highly processive polymerase may bebound on or near a biosensor adapted to measure the incorporation ofnucleotides.

In some embodiments, the method sequences a single DNA molecule byattaching a polymerase enzyme to a biosensor in a volume and causing aDNA template with associated primers to enter the volume and hybridizeand be held by (or in proximity to) the polymerase. Subsequently, thesequence may be determined upon extension of the primers by thepolymerase.

In yet other aspects, the invention provides a chamber-free device,comprising: an electromagnetic sensor array, a magnetic carrier forcarrying or holding molecules of interest at or near the electromagneticsensors, and a mechanism for removing the magnetic carrier via liquidflow and/or electromagnetic removal. The electromagnetic sensor may beone of a nanoneedle or a nanobridge, and the device may further compriselocal amplifiers. In some embodiments, the electromagnetic sensor has anarrow structure, and is etched under the structure such that both sidesof the sensor's surface are accessible to changes in pH, or to changesin conductivity. The devices and methods described herein may includeone or more improvements including incorporating materials having areduced zeta potential, using reagents that allow for bothpolynucleotide incorporation and sensitive pH measurements, and designand fabrication of nanosensors in optimal proximity and configurationrelative to a bead or substrate holding polynucleotide templates for asequencing reaction.

Other aspects and embodiments of the invention will be evident to one ofskill in the art based on the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a complete integrated system, along with schematic detailsof some subsystems. FIG. 1B shows schematic details of a sample andlibrary prep subsystem. FIG. 1C shows schematic details of a DNAfragmentation and purification subsystem. FIG. 1D shows a PDMS librarypreparation subsystem.

FIG. 2 shows a magnetic and virtual confinement array.

FIG. 3 shows a Comsol simulation the electric fields for a virtual well.

FIGS. 4A-4D show a schematic, drawings and a fabricated PDMS valvingsubsystems.

FIG. 5 shows an embodiment for a combined NanoNeedle sensor and magneticarray element.

FIG. 6 shows two versions of magnetic arrays which may position magneticbeads in fixed locations.

FIG. 7 shows an embodiment of a magnetic array which may locate beads ina fixed location.

FIG. 8 depicts schematically an element of an array utilizing a “leakyvalve” to localize a bead.

FIG. 9 depicts a simulated voltage and current plot associated with aredox reaction for detection.

FIG. 10 illustrates the charge distribution of a DNA binding protein.

FIG. 11 shows a schematic depiction of a magnetic array utilized forplanar magnetic particles.

FIG. 12 schematically illustrates a combined magnetic, virtual well, andNanoNeedle array element with a bead.

FIGS. 13A-13E show drawings, illustrations, and photomicrographs ofvarious enrichment module embodiments.

FIG. 14 illustrates the bead loading density for an existing flow cell.

FIG. 15 schematically illustrates the valving system and interfaces fora multichannel flow cell with proximate valving control.

FIG. 16 schematically illustrates a simple model for the impedances in aNanoNeedle and bead array element.

FIG. 17 schematically depicts an under-etched stacked NanoNeedle.

FIG. 18 is a photomicrograph of an array of under-etched stackedNanoNeedles.

FIG. 19 schematically depicts a 2D array of under-etched stackedNanoNeedles.

FIG. 20 is a photomicrograph of a 2D array of under-etched stackedNanoNeedles.

FIGS. 21A-C schematically depicts an element of an array of a singleside contact NanoNeedles.

FIGS. 22A-D schematically and diagrammatically show an element of anarray of a double sided contact NanoNeedle.

FIG. 23 schematically depicts the elements of a NanoBridge.

FIGS. 24A-C schematically depicts views of a ring NanoBridge.

FIG. 25 schematically depicts am array of NanoNeedles utilized forsingle molecule sequencing.

FIG. 26 illustrates sequencing data and the linearity of same from aNanoNeedle array element.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for methods and systems for DNAsequencing, and other types of biological or genetic analysis. Theinvention provides methods and systems for sequencing clonal DNApopulations or arrays of single molecule DNA, including by electronicsequencing, thereby providing a low cost and convenient sequencingplatform. In some aspects, the invention provides methods that monitorfor and/or correct for phase error during sequencing clonal populationsof DNA, to thereby improve accuracy and read lengths. Alternatively, theinvention provides methods and sensors for sequencing single moleculesof DNA, to thereby avoid such phase errors. In other aspects, theinvention provides arrays, including magnetic arrays, and virtualreactors for highly parallel reactions. These systems in someembodiments include nanosensors for detecting biological reactions orinteractions, including incorporation of nucleotides during DNAsequencing. Further, the invention provides integrated systems foramplifying and sequencing DNA samples.

Monitoring and Correcting for Sequencing Phase Errors

As used herein, “phase error” is defined as the occurrence where sometemplate polynucleotides of a clonal population are extended more orless than the consensus state. For fragments where a base is added whereit shouldn't be added relative to the consensus, this phase error isconsidered to be “leading.” For other template molecules where a base isnot added where it should be added relative to consensus, thepolynucleotide is considered to be “lagging.” Since polymerases areimperfect, some phase error is inevitable within a colony that has along extension reaction as a part of a colony based sequencing process.Phase error limits the read lengths of commercial clonal sequencingsystems.

“Leading sequencing incorporation error” refers to sequences that getahead of the dominant sequence through incorrect additions ofnucleotides. The incorrect additions may result from polymerase errors,particularly when high concentrations of dNTPs are used in anoncompetitive reaction. Alternatively, the leading sequencingincorporation error may result from inadequate washing or nonspecificbinding of dNTPs, which may be subsequently released and incorporated.“Lagging sequencing incorporation errors” refers to sequences that getbehind the dominant sequence through missed additions of the correctnucleotide; this may occur due to non-optimal reaction conditions,steric hindrance, secondary structure, or other sources of polymeraseinhibition. Longer cycle times can allow more opportunities for thepolymerase to incorporate the wrong nucleotide. Similarly lessaccessible DNA may result in inadequate opportunities to incorporate thecorrect nucleotide. It is anticipated that temperature, step times,polymerase selection, nucleotide concentration, salt concentration andbuffer selection may be optimized to minimize incorporation errors.

For example, a DNA sample may have a sequence of TGTTC in a first regionafter a region which is complementary to a primer. A fluidic cycle mayfirst introduce dCTP, secondly followed by dTTP, thirdly followed bydATP, and fourthly followed by dGTP, interspersed with wash steps. Inthe first part of a fluidic cycle, dCTP molecules which flow in as partof said first cycle may not be properly washed out of a well structure.In a second part of a fluidic cycle, dTTP molecules which flow in aspart of said second cycle may not be properly washed out a wellstructure. During the first and second part of the first fluidic cycle,no dNTPs should be incorporated. During a third part of a fluidic cycle,dATPs may be introduced and may be incorporated, as dATP iscomplementary to T, the first base of the sample. Any nonspecificallybound dCTP molecules which cease to be nonspecifically bound may also beincorporated during this third portion of a fluidic cycle. These unbounddCTP molecules may be incorporated after a dATP molecule isincorporated. After a dCTP molecule is incorporated, two more dATPmolecules may subsequently be incorporated, which may result in some ofthe molecules of a monoclonal bead having leading sequencing phaseerrors. Thus some molecules of a monoclonal bead may become “out ofphase”.

When a polymerase is provided with a single nucleotide or nucleotideanalog at a time, the error rate is typically significantly higher thanwhen all four nucleotides or nucleotide analogs are provided. This mayoccur despite the enormous difference in the catalytic efficiencymeasured as k_(poi)/K_(d,app), which may be four logs or more lower fora mismatched nucleotide vs. a matched nucleotide. Most of this is due tothe difference in K_(d,app). For example, Klenow polymerase has amisincorporation rate of one base in every 10⁶ to 10⁸ bases. Incomparison, polymerase extension reactions by current commercial systemsutilizing the incorporation of single native dNTPs may be limited to 100to 1000 bases. The polymerase in these systems spends almost all of itstime trying to misincorporate bases, leading to significant “leading”phase errors. Alternatively dephasing may result from a polymerase notincorporating a base in an incorporation fluid flow cycle due to theabsence of said polymerase, followed by the presence of a polymerase ina subsequent incorporation fluid flow cycle, or from a sufficiently lowcombination of dNTP concentration and time for incorporation such that abase is not incorporated, resulting in “lagging” phase error. Even whena nucleotide added to the system is the next nucleotide to be added, thereaction time must be long enough to complete the reaction for ahomopolymer, which may be eight or more nucleotides, or for DNA strandsthat may be less accessible from steric hindrance.

In one aspect, the invention provides methods for parallel or clonalpolynucleotide sequencing. In certain embodiments the method comprisessequencing a first portion of a template polynucleotide population, andcorrecting for phase error. Sequencing then continues to a seconddownstream portion of the target polynucleotide population. In variousembodiments, the sequencing may involve one or more of clonal sequencingof an array of polynucleotide populations (e.g., a bead array),electronic detection of nucleotide incorporation, and an electronic wellto isolate or concentrate sequencing reaction components. In variousembodiments, the invention provides methods for monitoring for andcorrection both leading and lagging phases, and the various approachesdescribed herein may be used individually or in any combination.

As used herein, “clonal” means that substantially all of the populationsof a bead or particle may be of the same template nucleic acid sequence.In some embodiments there may be two populations associated with asingle sample DNA fragment, as would be desired for “mate pairs,”“paired ends”, or other similar methodologies; the populations may bepresent in roughly similar numbers on the bead or particle, and may berandomly distributed over the bead or particle.

In some embodiments, the colony is re-phased by providing sequencing byincorporation nucleotides in different orders than might be otherwisenormally done. For example, if a system predominately has lagging phaseerror (as opposed to leading phase error), with for example a simple 1%lagging error per base (and all four different bases have similarlagging error rate), after 20 bases have been incorporated, just over75% of the members of the colony may be in phase, while over 20% may belagging by a single base. By the time 70 bases have been sequenced, lessthan half of the members of the colony will be in phase, 35% will belagging by a single base, 13% will be lagging by two bases, and 3% willbe lagging by three bases. So, for the following exemplary incorporationsequence example where the ideal position is shown in bold ( . . .CGATCGATCGA), 50% of the colony will be in phase at the fourth base (T),35% will be lagging one base (A), 13% will be lagging two bases (G), and3% will be lagging three bases (C). If the previous order forincorporation of the bases had been CGAT, and a C is provided, thelagging error will continue, and will be slightly increased. If insteadC is excluded the next base provided is G, the leading base will not beextended, while the portion of the colony which is lagging two bases atthe first G shown will be extended; if an A is provided next, most ofthe colony will now be in phase. If a three base combination without C,for example GAT is provided one or more times, any phase error will beconcentrated at C bases. Statistically some sequences may become moreout of phase as a result, but most sequences may be made to be more inphase. Two base combinations may also be used in much the same manner tore-phase the colonies, and a mixture of two base and three base sets maybe used. After re-phasing, the system may revert to four basecombinations and rephasing can be repeated as frequently as necessary.

In other embodiments, four base combinations may not be used at all, butalternating three and two base sets are used exclusively. In a furtherembodiment, the four bases are added in any combination of three and twobase sets of nucleotides, with the composition of the two and three basesets alternating in some embodiments. In some embodiments, said basesets described may also include the use of unincorporable nucleotides.In other embodiments, the concentrations of the nucleotides and orunincorporable nucleotides utilized in two, three or four basecombinations may vary from cycle to cycle, or from set to set.

In certain embodiments, phase error is corrected by excluding at leastone nucleotide base from a sequencing reaction. For example, phase errorcan be corrected by adding a combination of three nucleotide bases,thereby pausing each nascent polynucleotide in the clonal population atthe first occurrence of the excluded nucleotide base.

In certain other embodiments, phase error is corrected by reversiblyincorporating, into the in-phase polynucleotide strand, a chainterminating nucleotide. Once lagging phase strands have caught up to thein-phase strand, the terminating nucleotide is removed. This approachmay be most advantageous when the sequence being sequenced includes ahomopolymer region. For example in the following sequence fragment . . .AGCTCCC, where the in phase portion of the colony has incorporated the Tbase, with most of the lagging sequence having incorporated the C, G andA bases as the final bases of the members of the colony, if a C′terminating nucleotide is provided, followed by the base combinationAGT, AGT, then there may be a predominantly bimodal population, wherethe sequences . . . AGC′ and . . . AGCTC′ predominate. Said terminatormay then be removed from the C′ nucleotides, and another C′ terminatingnucleotide may be provided, resulting in two predominant sequences: . .. AGC and AGCTCC′. The C′ terminated nucleotide may then be followed bythe base combination AGT, AGT, resulting in the two populations: . . .AGCT and . . . AGCTCC′. The terminator may then be removed, andnon-terminated C nucleotides may then be provided, resultingpredominantly in a single sequence: . . . AGCTCCC.

In some embodiments, phase error is anticipated at certain positions(e.g., homopolymeric regions) based on a reference sequence, thusallowing the phase correcting to be efficiently implemented at anappropriate place in sequencing.

These approaches may be most effective for those systems which have apredominate source of error, such as, for example, a lagging error. Basecombinations which may be used for re-phasing may be added individually,so that the complete sequence of incorporation may be determined, or maybe added together, so that re-phasing may be accomplished with a smallsection of missing data. In some embodiments, reversible terminators maybe used repeatedly during a sequencing process or method, and may becombined with incorporable or unincorporable nucleotides.

In yet other embodiments, phase error is corrected by adding one or moreoligonucleotide clamps, the clamps hybridizing to the targetpolynucleotide to halt the sequencing reaction, and thereby remove phaseerror. Such a clamp could be a PNA fragment, a DNA fragment, or othermolecule, native or non-native, which binds specifically to a sequenceof DNA. In a system which is utilized for targeted resequencing,specific oligos may be used as “clamps”. The “clamps” may be provided atthe same time that primer sequences may be provided, prior to whenprimer sequences may be provided, after primer sequences may beprovided, before any sequencing reactions have been completed, or aftersome sequencing reactions have been completed. Multiple differenttargeted or untargeted clamps may be provided for each template.

Said clamp(s) may be random or targeted to specific regions of a DNAtemplate. A DNA fragment or other clamp may be further stabilized by theuse of histones, cationic protamines, recombinase, and other moleculesknown to stabilize duplex DNA. Sequence reactions may then proceed up tothe point of the clamp(s). Additional incorporation reactions may beperformed, using single bases, two base combination, three basecombinations, or four bases simultaneously. Said clamps may bepositioned such that said clamps may be spaced such that (on average)from ten to fifty bases exists between the 3′ end of the primer and saidclamp(s), or may be positioned such that (on average) ten to 100 basesexists between the 3′ end of the primer and said clamp(s), or may bepositioned such that (on average) 100 to 500 bases may exist between the3′ end of the primer and said clamp(s), or may be positioned such that(on average) 300 to 500 bases may exist between the 3′ end of the primerand said clamp(s), or may be positioned such that (on average) 1000 to5000 bases or more may exist between the 3′ end of the primer and saidclamp(s), or may be positioned such that (on average) 2000 to 5000 basesmay exist between the 3′ end of the primer and said clamp(s).

In some embodiments, the clamp may have a specific number of bases whichmay specifically hybridize, and may have additional bases which mayserve to stabilize said clamp. If the sequence of said clamp is nottargeted to a specific region(s), but is instead a non-targeted clamp,the sequence of the clamp may be selected using several criteria,including the stability of the clamp, the frequency of the selectedclamp sequence in the genome of interest, or in genomes of a similarnature, or in a chromosome of interest, or in a transcriptome ofinterest. The hybridization stability of the complete clamp, includingany non-specific bases such as deoxyinocine, 5-nitroindole, or abasicnucleotides, or may include any of the universal bases described in U.S.Pat. No. 7,575,902, which is hereby incorporated in its entirety byreference, and may include the stability of the bases selected asspecific bases for the clamp.

In some embodiments, said clamps may comprise 5, 6, 7, 8, 9, 10 or morespecific bases. Said clamps may be used for a number of DNA colonies,wherein substantially all of the colonies may have different DNAsequences from other DNA colonies. In some embodiments a single clamptype, comprising a single set of specifically hybridizing dNTPs may beused. In other embodiments, multiple clamp types, wherein the number ororder or spacing of specifically hybridizing bases may be different. Forexample, two different hexamer clamps may be used to decrease theaverage spacing, as measured in DNA bases, from the primer to the clamp,or between one clamp and the next clamp, that which would occur if onlyone of the two hexamer clamps were utilized, but may be larger than thatwhich might occur were a single pentameric clamp to be used. In someembodiments, the spacing, as measured in DNA bases from the primer toclamp, or from clamp to clamp may be varied as a result of the choice ofthe sequence of the clamp, as there is significant variation (more than20×) in the representation of different hexamers in the transcriptome(Anderson et al RNA V14(5)).

In some embodiments, the clamp(s) are subsequently removed (afterphasing) by raising the temperature, changing the pH or ionicconcentration, resulting in the denaturation of the clamp, but leavingthe longer extended primers, which may subsequently be further extendedafter the removal of the clamp(s). In other embodiments, the clamp(s)may comprise a nick site(s) which may be subsequently nicked by anappropriate nickase or endonuclease, which may destabilize the clampsufficiently to denature it. In some embodiments, the clamp may comprisecleavable linker sites, where said cleavable linker site(s) may bechemically cleavable or photocleavable. In some embodiments, a baseterminated at the 3′ position may be provided as a part of the clamp(s).Said terminators may be subsequently removed after nucleotides have beenadded so as to effect rephasing. Said terminator may be removed usingchemical or photochemical processes. In some embodiments, a combinationof different types of cleavable linker sites (e.g., unique cleaablelinker sites) are used for different clamps, so that the clamps withdifferent linker types may be provided prior to beginning anysequencing, or after sequencing has commenced, and different cleavablemechanism may be used to denature the clamps in an order which permitsmultiple rephasing of the template DNA.

In some embodiments, the method comprises adding clamps after asequencing and rephasing process has occurred. In further embodiments,the process of adding additional clamps may be repeated multiple times,such as 2 to 5 times, 4 to 10 times, or more than 10 times.

In some embodiments, data may be collected as incorporations may beperformed for all bases preceding a position adjacent to said clamp; inother embodiments, data may not be performed for all bases preceding aposition adjacent to said clamp.

In some embodiments, said clamps may be utilized in combination withnon-strand displacing polymerases, such that when said polymerasereaches said clamp through a polymerization process, said polymerasecannot displace said clamp. In further embodiments, the 5′ base of saidclamp may be linked utilizing a non native linker which cannot becleaved by a 5′ to 3′ exonuclease activity which said polymerase mayhave. In an alternative embodiment, said polymerase may be a non-stranddisplacing polymerase and may further be lacking 5′ to 3′ exonucleaseactivity.

In a further embodiment, a strand displacing polymerase may be used incombination with a clamp which is resistant to strand displacement bysaid strand displacing polymerase. Said clamp may consist of,particularly at the 5′ end of the clamp, non native bases that areresistant to the strand displacement activity of a strand displacingpolymerase. Such a base may comprise an abasic base, such as a basewhich has been depurinated, or synthetic bases such as PNAs, arabinosylderivatives of nucleobases, ribonucleotides, 2′-O-alkylribonucleotides,2′-O-methylribonucleotides, or bases with methylphosphonate linkages.

In some embodiments, the clamp, after phasing, is used as a primer. Saidclamp may include a reversible terminator at its 3′ terminus, where theprimer extension reaction proceeds until the clamp substantiallyprevents further extension. Further extension may be followed be theremoval of the terminator from the 3′ terminus of the clamp, permittinga polymerase to initiate a primer extension reaction from saidclamp/primer.

In some embodiments, a single clamp/primer is used for a colony or a setof colonies wherein the distance between said primer and said clamp maybe significantly more than the average sequencing length beforedephasing normally would occur, for example, when it is desirable to usesaid clamp(s) for the purpose of determining the structure of the DNAe.g. creating a scaffold and removing sequence ambiguity due torepetitive sequences. In some embodiments, the distance between theprimer and the clamp/primer may be twice as long as the average sequence“read length” before dephasing, or may be from twice as long to fivetimes as long as the average sequence “read length” before dephasing, ormay be from five times as long to twenty times as long as the averagesequence “read length” before dephasing, or may be from twenty times aslong to fifty times as long as the average sequence “read length” beforedephasing. Additional clamp/primer(s) may optionally be utilized in thisembodiment to extend the read length, and or elaborate further thestructure of the DNA colony(s). In some embodiments, the average readlength is about 50 nucleotides, or about 100 nucleotides, or about 200nucleotides, or about 300 nucleotides, or about 400 nucleotides, orabout 500 nucleotides. In other embodiments, a clamp/primer is used fora colony or a set of colonies where the distance between said primer andsaid clamp/primer or from the clamp/primer to the next clamp/primer maybe similar to the average sequencing length before dephasing normallywould occur, for example, as would be desirable to extend the length ofread of the average sequencing length.

If there is any variation in the stopping point of incorporation as aresult of interactions between the clamp, including any stabilizingmoiety, and the polymerase (or ligase), a clamp re-phasing method may becombined with one of the methods previously described, which may beadvantageous as the sequence of the clamp is already known, permittingaddition of bases other than the first base of the clamp sequence,potentially followed by bases other than the second base of the clampsequence, or any stopping at any other known portion of the clampsequence.

In order to permit short hybridization probes as rephasing reagents,stabilizing compounds such as hydralazine, or antitumor antibioticcc-1065 may be employed. Similarly the probe may be a PNA or LNA probe,which may provide the dual function of providing tighter binding, andprecluding the need to prevent the probe from being extended bypolymerase, by using for example, a terminator at the 3′ end of theprobe. Additionally the probe may be a single plex, a duplex which mayhybridize to the target DNA to create a more stable triplex, or may be atriplex, which may hybridize with the target DNA to form a quadraplex.In some embodiments the probe may be provided in two or more pieces,wherein one portion may be a hybridizing single plex, and a secondportion may be hybridize to create a triplex. In some embodimentsadditional portions to the probe complex may be provided, allowing theformation of a quadraplex, or the formation of a duplex with more thantwo pieces in addition to the original template.

In other embodiments, lagging dephasing may be reduced by “stockpiling”polymerase enzymes on or near the DNA which is to be extended andsequenced, such that a number of polymerases may be available for eachactive polymerization site. Said stockpiling may result from nativebinding of the polymerase to the DNA. Such binding may result normally,as for example when a Klenow polymerase is used, where the Klenowpolymerase has intrinsic ssDNA and dsDNA binding.

Alternatively, in some embodiments, the DNA may be provided with 3′terminated random primers in addition to universal or targeted primers,where said universal or targeted primers may be not terminated, andwhere said polymerase may bind at the 3′ terminated end of said randomprimers, as well as to the 3′ end of said universal or targeted primers.As said random primers may be terminated, said random primers may not beextended, and thus may not contribute to the signal concomitant toextending the strand from said universal or targeted primers. In thisembodiment, the polymerase may be lacking in 3′ to 5′ exonucleaseactivity, such that said random primers may be not degraded, resultingin a loss of stockpiling capacity.

In an alternative embodiment, random primers using nucleotide analogs inat least the 3′ terminus may be employed instead of 3′ terminated randomprimers, where polymerase will bind to the random primers, but will notextend them. In this embodiment, the polymerase may have 3′ to 5′exonuclease activity if said 3′ to 5′ exonuclease activity iseffectively inactive in removing the nucleotide analogs. In someembodiments, it may be desirable for the K_(d) to be smaller for apolymerase binding to said 3′ terminated random primers, or randomprimers having one or more nucleotide analogs in the at least 3′terminus position. Said nucleotide analog containing random primers maybe chimeric, where said chimera comprises native nucleotides andnucleotide analogs, multiple types of nucleotide analogs, or nativenucleotides and multiple types of nucleotide analogs.

In a further embodiment, the random primers may be 3′ terminated randomprimers, wherein the 3′ terminus of the random primers further comprisesa thiophosphate nucleotide in the 3′ (terminated) position, such thatthe random primers are further resistant to 3′ to 5′ exonucleaseactivity. The 3′ thiophosphate primers may be commercially availablefrom, for example, IDT (Integrated DNA Technologies). In thisembodiment, native polymerases with 3′ to 5′ exonuclease activity suchas phi29 may be used, without needing to mutate the polymerase toinactivate the exonuclease activity to prevent degradation of saidrandom primers. Such thiophosphates may be alpha-S or alpha-Rsteroisomers. The random primers may also comprise 5′ thiophosphates,such that 5′ to 3′ exonuclease activity may be inhibited. Alternatively,the random primers comprise 3′ inverted dT, which may act to preventboth polymerization and exonuclease activity with respect to the 3′position of the random primer. Dideoxynucleotides may be used asterminators. Said terminators may be reversible terminators, virtualterminators, terminators attached to the base of the nucleotides, or toany position of the sugar the nucleotides. The nucleotides in the randomprimers may be natural bases, or may be a synthetic bases. Said randomprimers may comprise dNTPs, or may comprise chimeras in combination withPNA, RNA, LNA, 5-Nitroindole, deoxyInosine, or other non-natural dNTPs.

Stockpiled polymerases may also be bound to the surface of a bead, tothe surface of a sensor, to interstitial regions between sensors, togroups, linkers, or polymers attached to said beads, sensors orinterstitial regions. Such binding may be to additional strands of nonextendable exonuclease resistant DNA, synthetic DNA or other linearpolymers, or may be other binding groups such as antibodies, wherein thebinding groups may bind directly to said polymer, or may bind to anintermediate protein which may complex with said polymerase.

The relationship in the relative kinetics between the K_(off) of thepolymerase from the active incorporation site, and the K_(off) stockpilesite(s) and the number of stockpiled polymerases, and the extensionperiod must be appropriate in order to insure that a stockpiledpolymerase will be able to replace a polymerase which has disassociatedwith the active incorporation site of a DNA strand in order toincorporate a base(s) and prevent lagging dephasing within a desirederror rate. For example, if the K_(off) is the same for both the activeincorporation site, and the stockpile site(s) K_(off) and K_(on), andthe K_(off) is equivalent to 20 incorporation fluidic cycles, if one had20 stockpiled polymerases, the odds that another polymerase will becomeavailable to bind to the active incorporation site is less than 50%.This can be improved by reducing the K_(off) of the stockpile sites, andincreasing the K_(on) of the Stockpile sites, with the caveat that lossof polymerase to fluid flow may be an issue. The Koff may be reduced asa result of utilizing non natural bases, terminators, or the associationof proteins which may normally reduce the processivity of the polymeraseto the binding site.

Alternatively or in addition, the polymerase may be a single type ofpolymerase, or may be a combination of different types of polymerases.In general, commercialized more processive polymerases have lowerincorporation error rates, and as such it may be desirable to mainly usehighly processive polymerases. It may be desired to have one type ofpolymerase have a significantly longer K_(off) than another type ofpolymerase. It may also be appropriate to have more shorter Koffpolymerase available to replace any more highly processive (longerK_(off)) polymerase that disassociates, such that a polymerase will beavailable to incorporate any bases as appropriate. As such, it may beappropriate for the Koff of the less processive to be less than the timeperiod allocated for an incorporation fluidic cycle, in order to insurethat polymerases will be available for incorporation should a moreprocessive polymerase become disassociated from the binding site of theextending primer(s).

In some embodiments where it is preferred to use two or more types ofpolymerase, it may be desirable to preferentially bind a more processivepolymerase to the primer which is to be extended. It may thus bedesirable to allow a more processive polymerase to bind to the primerwhich is to be extended prior to adding a less processive polymerase,which may have ssDNA and or dsDNA binding moieties associated therewith.In other embodiments, it may be desirable to modify or mutate apolymerase such that a binding moiety may be added to said polymerase,such that the polymerase may bind directly to portions of ssDNA ordsDNA.

The stockpile of polymerases may be replenished periodically. Saidreplenishment may occur with every incorporation cycle, or after anappropriate number of incorporation cycles have occurred. The number ofcycles between replenishment may vary depending on the stockpilingmethod. For example, if the stockpiling method is storage on randomprimers, the number of stockpile locations reduces with the extension ofthe universal or targeted primer, as said random primers may bedisplaced by the polymerase, using either strand displacement, or 5′ to3′ exonuclease digestion of the random primers. The number of stockpiledpolymerases may be maintained, if for example, a second bindingmechanism exists for binding to double stranded DNA.

In certain embodiments, the method comprises monitoring the reaction forloss of signal, and rephasing to restore sequencing signal. For example,data from a sequencing reaction may be monitored, and rephasing may beperformed when it is observed to be needed, as for example, as seen bysignal levels which may be less than expected for a single base, butwould be expected if lagging phase error were present, or by thereduction of the signal level observed for single bases. Saidobservations may consider nominal sequence in determining whether asignal would statistically result from lagging phase error. Saidobservations may include a histogram of the signal levels for asequencing fluidic cycle or a set of sequencing fluidic cycles.

In some embodiments rephasing may be performed for any clonal sequencingsystem, including those which utilize four incorporable nucleotides, aswell as all of those described above with respect to minimizingdephasing. In some embodiments, the rephasing is performed in connectionwith emulsion PCR, or alternatively, the magnetic or bead arraydescribed herein, optionally in connection with electronic sequencing.

In one embodiment of the current invention, compensation may beperformed to reduce for phase error by using earlier and/or later datato determine expected background levels for each cycle for eachlocation. Expected phase error for each base, for each base in thesequence context, and the amount of lagging and leading error previouslydetermined may be used to assist in determining the actual base. Thiserror correction may also take into account phase errors fromneighboring reactions on an array, as well as the influence of neighborson the signals received from each sensor.

In some embodiments the distribution between leading and lagging phaseerror is influenced such that one type of phase error may occur at ahigher rate than the other type of phase error. In one embodiment theconcentration of dNTPS may be limited, so that lagging phase error ismore likely than leading phase error. In a further embodiment, rephasingmay then be performed to correct for the more probable phase error type.In other embodiments, the method acts to correct for both types of phaseerrors, wherein the method corrects for one phase error type, and thencorrects for the other type of phase error. Said method(s) for phasecorrection may be repeated periodically through the sequence process. Insome embodiments, the fluidic pattern for rephasing may be fixed. Forexample, the fixed pattern may have a fixed number of fluidic sequencingcycles between performing rephasing methods, or the number of fluidicsequencing cycles may change during the sequencing process, for example,by reducing the number of fluidic sequencing cycles between performingrephasing methods.

In another aspect, the invention provides a method for reducing leadingphase error in parallel or clonal polynucleotide sequencing. The methodaccording to this aspect comprises sequencing a population ofpolynucleotides in the presence of a competitive reaction. Thecompetitive reaction comprises either nucleotide bases or nucleotidederivatives for all four nucleotide bases, wherein three of the fournucleotide bases are unincorporable into the growing polynucleotidestrand. The sequencing may involve one or more of clonal sequencing ofan array of polynucleotide populations (e.g., a bead array), electronicdetection of nucleotide incorporation, and an electronic well to isolateor concentrate sequencing reaction components. The unincorporablenucleotide in various embodiments may be selected from (withoutlimitation) a PNA nucleotide, a LNA nucleotide, a ribonucleotide, anadenine monophosphate, an adenine diphosphate, an adenosine, adeoxyadenosine, a guanine monophosphate, a guanine diphosphateguanosine, a deoxyguanosine, a thymine monophosphate, a thyminediphosphate 5-Methyluridine, a thymidine, a cytosine monophosphate, acytosine diphosphate cytidine, a deoxycytidine, a uracil monophosphate,a uracil diphosphate, a uridine, and a deoxyuridin. Generally, theunincorporable nucleotides are bound by the polymerase, but are notincorporated into the growing polynucleotide strand by the polymerase.In some embodiments the concentration of the unincorporable nucleotidesis relative to the polymerase activity for each of the unincorporablenucleotides. Said unincorporable nucleotides or nucleotide analogs maybe unlabeled, optically labeled, or charge labeled.

In some embodiments, the concentration of the incorporable dNTPs may beincreased relative to the concentrations of incorporable dNTPs,permitting a decrease in both the leading and lagging error rates.

In still another aspect, the invention provides a method for reducinglagging phase error in a population of polynucleotide templates. Themethod comprises stockpiling polymerase enzyme on or near the targetpolynucleotide during a sequencing reaction, such that polymerase isavailable for each or substantially each active polymerization site.

Alternatively or in addition, the method comprises binding a repairprotein or single stranded DNA binding protein to the targetpolynucleotide to, among other things, remove secondary structure or aidprocessivity. In some embodiments, it may be desired to reduce secondarystructure of single stranded DNA which may otherwise interfere withpolymerase activity, resulting in lagging phase error. In oneembodiment, a protein which binds to single stranded DNA is used. Suchproteins may include repair proteins such as bacterial RecA DNA repairproteins, HIV nucleocapsid proteins, T4 bacteriophage gene product 32,calf thymus UP1, Epstein-Barr virus BALF2, or commercialized singlestranded binding proteins such as Epicentre E. coli single strandedbinding protein, as well as many others. In some embodiments, saidsingle stranded binding proteins may also serve to aid the processivityof the polymerase. In other embodiments other moieties may also aid inthe processivity of the polymerase, such as Epstein-Barr virus BMRF1,triplex S. Cerevisiae proliferating cell nuclear antigen, T4bateriophage gene product 45, thieoredoxin, E. coli PolIII holoenzyme,eukaryotic clamp protein PCNA, or other DNA sliding clamp proteins, orother double stranded or single stranded DNA binding moieties.

In some embodiments, polymerase processivity may be enhanced bymutations to the polymerase, such as the addition of ahelix-hairpin-helix domain to Phi29 polymerase, or the use of achimerical polymerase as described by Salas et al in PNAS 107 16506, theaddition of a thioredoxin binding domain, the addition of an archaealsliding clamp, DNA binding protein Sso7d, a zinc finger domain, aleucine zipper, amongst other possibilities. In some embodiments, thepolymerase may be further modified such that more than one processivityenhancement mutation is used, such as mutations to add both doublestranded and single stranded binding moieties at the respective ends ofthe polymerase. In some embodiments, such binding moieties are alsoindirectly bound to the polymerase to, for example, add a streptavidinmoiety by mutating a polymerase, and adding via mutation a biotin moietyto a single stranded DNA binding moiety and or double stranded DNAbinding moiety such as those mentioned above. The polymerase will thusbe bound to the single or double stranded DNA binding moiety through thestreptavidin biotin binding, and further bound to the DNA through saidsingle or double stranded binding moiety. In other embodiments, otherbinding moieties are employed to bind a polymerase to a single strandedor double stranded DNA binding moiety, wherein an additional moiety maybe added via mutation to each of the polymerase and the single or doublestranded binding moiety, wherein the additional moieties added to thepolymerase and the single or double stranded binding moiety have amutual binding affinity. In further embodiments, a moiety may be addedvia mutation to one of the polymerase, a single stranded DNA bindingmoiety and a double stranded DNA binding moiety, wherein the moietyadded via mutation may then bind to another of a polymerase, a singlestranded DNA binding moiety and a double stranded DNA binding moiety,such that a polymerase is bound to a single or double stranded DNAbinding moiety.

In some embodiments, the stockpiling is achieved by the use of thepolymerase's binding to non-extendable primers. In certain embodiments,the non-extendable primers are not subject to 3′ exonuclease activity ofthe polymerase. The non-exendable primers in some embodiments are 3′terminated random primers, and the extension primers are universal ortargeted primers. A polymerase binds at the 3′ terminated end of therandom primers, as well as to the 3′ end of the universal or targetedprimers. For example, the 3′ terminated primers may comprise athiophosphate nucleotide in the 3′ terminated position, such that said3′ terminated primers are resistant to 3′ to 5′ exonuclease activity.

The sequencing according to this aspect may involve one or more ofclonal sequencing of an array of polynucleotide populations (e.g., abead array), electronic detection of nucleotide incorporation, and anelectronic well to isolate or concentrate sequencing reactioncomponents.

In connection with electronic sequencing, in some embodiments it may bedesirable to use ion concentrations that are lower than might be optimalfor synthesis, in order to have an ion concentration sufficiently lowfor improved operation of the detector. This may result in phasingerrors, and a shorter sequence length than desired. It may be desirableto have sequence lengths longer than possible with said low ionconcentrations. Thus, in one embodiment, the effective read length isincreased by alternating conditions optimal for detection, withconditions optimal for synthesis. For example, the method may compriseperforming a sequencing reaction to the full length possible while usinglow ionic concentrations needed for optimal reading of said DNAextension reaction, melting off the extended primer strand, introducingnew primers and dNTPs, and proceeding with the synthesis reaction whileusing optimal ion concentrations for synthesis. The process of meltingoff the extended primer strand, introducing new primers and dNTPs, andproceeding with the synthesis reaction while using optimal ionconcentrations for synthesis, followed by changing the conditions tothose appropriate for detection may be repeated multiple times, untilthe process no longer results in useful data. As the determination ofhow many synthesis steps to use may be statistical, the process may bereversed, performing a synthesis with conditions optimal for synthesis,followed by performing synthesis using conditions appropriate fordetection; this may then be followed by melting off the extended primerstrands, introducing new primers, and using ionic concentrationsappropriate for detection.

In order to optimize detection sensitivity, ion and or dNTPconcentrations may be desirable which are below the concentrations whichmay be desirable for proper enzyme kinetics. This may result in longerincorporation times than desired, and/or more lagging phase error. Insome embodiments, it may be desirable to use more than one concentrationof dNTPs during a single incorporation cycle. For example, it may bedesirable to perform single incorporation cycles with low concentrationsof dNTPs and/or ions so as to optimize the sensitivity and signal tonoise of the sensors. Thus, a solution with a low concentration of dNTPsand/or ions may be flowed into a flow cell(s) so that measurements maybe taken. This may be immediately followed by a solution with aconcentration of dNTPs and/or ions which is optimal for incorporationinto the extended primer, such that minimal de-phasing may occur. In analternative embodiment, a high concentration of dNTPs may be flowed intoa flow cell(s), so that a quick and optimal incorporation reaction mayoccur, providing for minimal de-phasing. This may then be quicklyfollowed by a reagent solution with low or no dNTPs and an appropriatelylow ion concentration such that an optimal sensor reading(s) may betaken.

In another embodiment, a reagent solution with optimal concentrations ofdNTPs and/or ions for quick incorporation of nucleotides may be flowedinto a flow cell(s) to quickly incorporate a significant portion of thedNTPs into the various colonies as needed, followed very quickly byflowing into a flow cell(s) a reagent solution optimal for optimalsensor reading(s), followed by flowing into a flow cell(s) a reagentsolution with concentrations of dNTPs and/or ions at concentrationsappropriate for incorporation of nucleotides into the extended primersof the various primers. The first reagent solution may have aconcentration of nucleotides which may be sufficiently high that werethe reagent solution to be used for an extended time period, polymeraseincorporation errors might occur at a noticeable level.

Because polymerase efficiencies may be different for each base, it maybe desirable to use different concentrations of different dNTPs, usedifferent buffers, different cation concentrations, differentconcentrations of polymerase, different types of polymerase, or anycombination thereof to optimize the incorporation rate, minimize theamount of phase error, minimize the amount of incorporation error, andmaximize the read length.

In certain embodiments, the concentration levels of the differentnucleotides or unincorporable nucleotide analogs is matched to therelative polymerase activity for each of the nucleotide or nucleotideanalogs. For example dTTP binding rate has been measured to be differentby a factor of over two with respect to the other nucleotides. The otherthree nucleotides may be much closer in their polymerase binding rates,but still vary by over 10 percent with respect to each other. It islikely that the difference may be even larger in comparing thepolymerase binding rates for different unincorporable nucleotide analogsrelative to native nucleotides.

In a further embodiment, the concentrations of the unincorporablenucleotides needed for equivalent polymerase extension are higher,equal, or lower than the concentration for optimal primer extension withminimal dephasing for the one or more incorporable nucleotides ornucleotide analogs which may be provided for a sequencing reaction notutilizing unincorporable nucleotides or nucleotide analogs, making theprobability of misincorporation of nucleotides or nucleotide analogslower than if the concentrations of unincorporable nucleotides analogswere provided such that polymerase extension efficiency were matched, orif the unincorporable nucleotide analogs were provided at concentrationswith lower polymerase extension efficiency relative to the incorporablenucleotides or nucleotide analogs. Alternatively, the unincorporablenucleotide analogs may be provided at concentrations with lowerpolymerase binding rates relative to the incorporable nucleotides ornucleotide analogs, such that the reaction may proceed at a higher ratethan would occur if the polymerase binding rates of the unincorporablenucleotides were the same or higher than the incorporable nucleotideanalog binding rates.

In other embodiments, the concentration of the incorporable nucleotideor nucleotides provided may be varied such that the incorporation ratesfor the different dNTPs may be more equal. The concentrations may bemodified as needed for different buffer conditions, pHs, polymerases,and interactions with any polymerase clamp complexes, or other moietieswhich may be utilized to stabilize the polymerase.

In some embodiments, unincorporable nucleotides are used as part of areagent set which is not intended to cause the incorporation ofnucleobases. Such a reagent set may, for example, be intended to washout a previous set of incorporable nucleobases, prior to introduction ofa new set of incorporable nucleobases. Such a wash step may also includephosphatases to degrade triphosphate nucleobases to diphosphates ormonophosphates, so that any remaining triphosphates will be degraded andwill thus be unincorporable. The unincorporable nucleobases may occupy aposition complementary to the next base in the pocket of the polymerase,and may serve to effectively increase the processivity of thepolymerase, as the thumb of the polymerase will remain closed,attempting to incorporate the unincorporable nucleobase, and reducingthe disassociation of said polymerase from the DNA.

The above approaches may be used for reaction conditions where there maybe three unincorporable nucleotide analogs and one incorporablenucleotide or nucleotide analog, or where there are two unincorporablenucleotide analogs, and two incorporable nucleotides or nucleotideanalogs, or where there may be one unincorporable nucleotide analog, andthree incorporable nucleotide or nucleotide analog.

Detection methods which may be used with the above reaction conditionsand unincorporable nucleotide analogs may include any form of electronicsensing of incorporation or incorporation events, including ISFETs,CHEMFETs, NanoNeedles, NanoBridges, chemilumenescence detection,fluorescence detection, including detection of Qdots or othernonstandard fluorophores, and detection of intercalating fluorophores,detection using fluogenic moieties.

In further embodiments of the current invention employing bead arraysand electronic sequencing (as described herein), null beads or nullsensor regions (sensor regions which do not have associated colonies),may be used as references; such references may compensate for variationsin temperature, variations in the conductivity or pH of the bulkreagent, or localized variations in conductivity or pH. The control ofthe system will help limit and identify phase errors, thereby extendingread length.

A common practice for FET pH sensors is to use a reference electrode;some designs for arrays of FET pH sensors use a reference channel foreach detection channel; others have reference channels for a set ofdetection channels. But the local pH of the detector is influenced bythe presence of the DNA colony, and changes as the length of the secondstrand of DNA is extended by the polymerization reaction. In using achemistry whereby a single type of nucleotide is introduced to the flowcell at a time, many detector channels will not have a reaction takingplace at that detector; in fact most detector channels will not have areaction occurring. Thus in one embodiment, neighboring detectors areused as reference channels, providing the data analysis algorithms anopportunity to measure the pH or ion concentration as it changes indetectors which are neighboring detectors to a detector which has apolymerization reaction occurring. This permits detection of pH or ionconcentration levels, or other sources of noise local to the detector ofinterest, and may also permit detection of crosstalk, allowingmonitoring and modification of the crosstalk deconvolution function.

In certain embodiments, null beads (which do not have DNA colonies orwhich have colonies that do not have the same primer as that which willbe used for the sequencing by synthesis reaction) are used to insurethat some detectors will not have a polymerization reaction occurring atthem. Beads which have colonies of DNA with an appropriate primer may beintroduced to the flow cell, taking a random set of positions ondetectors. Subsequently, a set of null beads as previously described maybe introduced into the flow cell, whereby the null beads can occupyrandom locations not already occupied by beads already present in theflow cell. As reagents are introduced into the flow cell, the null beadsare then used to monitor the pH and or ion concentration levels,enabling the analysis algorithms to better determine background levelsand/or a crosstalk deconvolution function.

In another embodiment, null beads are introduced pair wise with samplebeads, and the signals are determined using a differential amplifier,obviating the need for the analysis algorithms to directly deconvolvevariations in background and crosstalk.

In still other embodiments, adjacent beads or sensor regions which donot have an incorporation reaction in the current fluid cycle may beused as a reference. As on average most beads or sensor regions will nothave an incorporation reaction when sequencing large populations ofdifferent sequences, these locations without an incorporation reactionmay be used as additional references. As references, beads or sensorregions without an incorporation reaction may also provide betterreferences relative to empty sensors or null beads, as DNA polymerase,and beads will be present in the volume of interest, and any variationin surface chemistry and resulting background counter ion concentrationwill likely be better matched. It is likely that different colonies onbeads or sensor regions may have colony DNA and/or extended primers ofdifferent lengths from the lengths of colony DNA and/or extended primersassociated with other beads and or sensor regions, and thus may havedifferent amounts of charge present which may interact with the sensor.

Thus in some embodiments of the current invention, software may need tocompensate for the relative length of DNA and/or extended primersassociated with sensors and the resultant different charge and signallevels, as well as the location of the beads or colony relative to thesensor, which may influence. The software may keep a record of thesignal levels: associated with each sensor prior to introduction of abead to said sensor; after introduction of a bead but prior tointroduction of a primer and or a polymerase(s); after introduction of aprimer and or a polymerase(s) but prior to introduction of a firstnucleotide in a sequencing by synthesis reaction; associated withcolonies without primers; associated with colonies which may havedifferent lengths of DNA; and the signal levels associated with colonieswith hybridized primers, which may have different lengths of DNA. Thesoftware may keep track of how many bases have been added to eachprimer. The signal levels may be an absolute level, or a relative levelbetween different sensors. The software may use a number of otheradjacent, or proximate sensors as references to determine the signallevel for an individual sensor, compensating for the length of the DNAfor each set of colonies, the length of the extended primers, and thesignal level which may be generated as a result of positioning of thebeads or colonies relative to the sensor. The software may alsocompensate for the relative position of the sensors relative to thetransition between a reagent volume which does not have dNTPs and areagent volume which does have dNTPs. The software may furthercompensate for variations in dNTP concentration as a function ofdiffusion and or dNTP concentration depletion, as a result ofincorporation of into extended primers. The amount of diffusion may becharacterized from earlier data from the same chip, which may be in thesame sequencing run, or from a previous sequencing run, or fromsequencing data from a previous chip utilized on the same instrument, orfrom data from other chips utilized on other instruments. The expectedlevel of depletion may be determined based on data generated as thetransition between a reagent volume which does not have dNTPs and areagent volume which does have dNTPs moves through a flow cell.

In some embodiments, control beads are used which have a known sequence,or with different known sequences. The known sequence(s) may havehomopolymer runs of different known length, and may be used to calibratethe response of the system to better determine the length of homopolymerruns of unknown length. The known sequences may also be useful indetermining the level of signal or background signal, as the length ofthe extended primer and whether an incorporation event has occurred willbe known in advance. The control beads may be used to differentiateinstrument problems from sample prep problems. The control beads withknown sequences may be generated outside of the system, and introducedwith colonies of DNA attached thereon, or control DNA may be mixed withor introduced prior to or after the sample DNA to generate DNA colonieson beads or otherwise associated with the sensors. In some embodiments,signal levels may be monitored and stored in a manner similar to thatdescribed herein for use with normalization from adjacent beads.

In a manner similar to optical aberration, the diffusion of speciesbeing detected by the detectors will cause crosstalk between differentdetectors. In one embodiment of the current invention, deconvolution ofdata taken from different sensors on the array may be performed in amanner similar to that used to deconvolve the point spread function froman optical system. The deconvolution function used may depend in part onthe temperature of the flow cell at the time of detection, as well asthe flow rate through the flow cell, which may tend to cause morecrosstalk “downstream” of a particular colony. The deconvolutionfunction which is utilized for said deconvolution may be a fixeddeconvolution function, or may be derived as part of a best fitalgorithm.

The sensor array may be made self calibrating, allowing calibration forsuch variables as amplification efficiency, bead size and loading, beadplacement on the sensor, etcetera. In general, in amplifying a DNAsample to create a monoclonal population of DNA on a bead, a firstprimer may be ligated to the sample DNA prior to said amplificationreaction. In the sequencing process, a second primer is provided whichis complementary to said first primer which has been ligated to said DNAsample. Said second primer may be several bases shorter than said firstprimer. Thus each monoclonal bead has a known initial sequence at adensity independent of amplification efficiency, which will be thesection of said first primer which is not matched by said complementarysecond primer. This may permit prior knowledge as to the base sequence,and may include calibration sequences such as known length homopolymerruns.

In an alternative embodiment, such calibration may occur after asequencing reaction is complete, or alternatively after a number ofbases have been sequenced in a sequencing reaction. Statistically, mostfluidic cycles in a sequencing reaction will not result in a baseincorporation at an individual bead. The next most common result from afluidic cycle will be the incorporation of a single base. Thus the dataset may be analyzed, and appropriate signal levels may be set for eachbead.

In a further embodiment, ongoing compensation/calibration may beimplemented as the signal level for a base incorporation in a fluidiccycle reduces during a sequencing process, and the background for a nothaving a base incorporation in a fluidic cycle as a result of factorssuch as loss of some of the clonal population on the bead, sequencingphase lead, or sequencing phase lag of some of the clonal population onthe bead or other factors. Thus a signal level may calculated as to howmuch signal may be expected for a fluidic cycle which has a single baseincorporation, no base incorporation, or multiple bases incorporated dueto a homopolymer run, at each point in a sequencing process.

In some embodiments, reverse phase alignment may be performed, wherein apolymerase with 3′ to 5′ exonuclease activity is used with a dNTP poolthat is missing at least one dNTP. The polymerase with 3′ to 5′exonuclease activity will remove bases back to the next dNTP in theprovided dNTP pool, at which point equilibrium will be reached, and nofurther nucleotides will be removed. This may be performed in order toremove any bases which have been incorporated due to leading phaseerror. In other embodiments, one or more base type may be incorporatedwhich does not permit exonuclease activity, such as a thiophosphatenucleotide. Exonuclease activity may then be used by the removal ofunincorporable nucleotides, improving the kinetics for exonucleaseactivity. In other embodiments, initial incorporations may performedwith an exo− polymerase, followed by the use a exo+ polymerase oranother nuclease to remove any bases back to a thiophosphate nucleotideor other nucleotide which is resistant to nuclease activity.

Single and/or Repeated Polynucleotide Sequencing

In another aspect, the invention provides a method for repeated and/orsingle polynucleotide sequencing. The method in this aspect comprisesproviding a circularized DNA sequencing template, and sequencing thetemplate by determining the sequence of incorporation of nucleotides bya DNA polymerase having 5′ to 3′ exonuclease activity. The sequencingaccording to this aspect may involve one or more of clonal sequencing ofan array of polynucleotide populations (e.g., a bead array), electronicdetection of nucleotide incorporation, and an electronic well to isolateor concentrate sequencing reaction components. In various embodiments,the DNA polymerase is highly processive and has reduced exonucleaseactivity. Further, the DNA polymerase may be bound on or near abiosensor adapted to measure the incorporation of nucleotides. In someembodiments, the method comprises pre-binding a polymerase to thepolynucleotide prior to sequencing. According to this aspect, theinvention avoids the need to correct phasing or re-phasing.

A single DNA molecule can be sequenced by a NanoNeedle biosensor (whichis described in detail herein). A polymerase enzyme is attached to thesensor. A DNA sample with associated primers may then be caused to enterthe volume with the polymerase attached sensors, using for example,pressure induced flow, electro-osmotic induced flow and or migration, orsimilar means. A single molecule from the DNA sample may then be boundby a polymerase attached to a sensor in a sensor array. Additionalsingle DNA molecules may also be bound by other polymerases bound toother sensors in the sensor array.

In order to permit repeated measurements of the same DNA sample, the DNAsample may be circularized, and the polymerase may be a stranddisplacing polymerase. Thus the DNA sample may be repeatedly sequencedby allowing the primer extension reaction to continue for many cyclescompletely around the circular DNA sample. The data for this strand canthen be converted into a more accurate consensus sequence with reduceddata processing. In a distinct advantage over a system which employsdetection of fluorophores, the system in this aspect uses the fullcapability of the read length of the polymerase, unhindered by havingthe read length reduced by phototoxicity.

A single molecule is a case of a monoclonal population in which thepopulation is 1. As such ideas that are relevant to monoclonal DNAtypically also apply to the single molecule condition situation.

FIG. 25 describes and illustrates a device and method whereby a singleDNA molecule 2507 can be sequenced by a NanoNeedle biosensor array 2500.A polymerase enzyme 2506 may be attached to a sensor 2501. A DNA samplewith associated primers may then be caused to enter the volume with saidpolymerase attached sensors, utilizing for example, pressure inducedflow, electrophoretic induced flow and or migration, or similar means. Asingle molecule from the DNA sample 2507 may then be bound by apolymerase attached to a sensor 2501 in a sensor array 2500. Additionalsingle DNA molecules 2507 may also be bound by other polymerases 2506bound to sensors 2501 in the sensor array 2500.

In one embodiment, one of the four native dNTPs 2502 is then flowed intothe channel volume 2504 with the sensors. If the dNTP is complementaryto the next base in the sample DNA 2507, it may be bound andincorporated. The NanoNeedle sensor 2501 may then detect the resultingchange in the local charge of the extended primer DNA, permittingdetection of the incorporation event, at each appropriate position ofthe sensor array 2500. If the sample has more than one base in a rowwhich is complementary to the type of dNTP 2502 which has beenintroduced into the channel volume 2504 with said sensors 2501, a secondor subsequent binding and incorporation of a dNTP 2502 may be detectedby said NanoNeedle sensors 2501. The dNTPs 2502 may then be washed outof the channel volume 2504 containing the sensors 2501.

In certain embodiments, one of the four native dNTPs is then flowed intothe volume with the sensors. If the dNTP is complementary to the nextbase in the sample DNA, it is bound and incorporated. The NanoNeedlesensor may then detect the resulting change in the local charge, whichmay be as a result of the change in charge of the extended primer DNA,or may be as a result of other charge changes, permitting detection ofthe incorporation event, at each appropriate position of the sensorarray. If the sample has more than one base in a row which iscomplementary to the type of dNTP which has been introduced into thevolume with said sensors, a second or subsequent binding andincorporation of a dNTP may be detected by said NanoNeedle sensors. ThedNTPs may then be washed out of the volume containing the sensors.

A different dNTP may then be flowed into the sensor array volume,permitting detection of incorporation events. Subsequent cycles ofwashing, introduction of each of the four dNTPs one at a time, anddetection of incorporation events permit determination of the differentsample DNA sequences.

In yet another embodiment, up to four different nucleotides may bedelivered simultaneously, and determination as to which nucleotide isincorporated may be determined by observation of the kinetics associatedwith the incorporation reaction.

In an alternative embodiment, the sample DNA may be bound to one of thepolymerase, the sensor, or a region between the sensors sufficientlyclose to the sensor that the bound polymerase may bind the sample DNAafter a primer has been introduced into the system and permitted tohybridize with the sample DNA. Subsequently, after completion of theprimer extension and associated determination of the sample DNAsequence, the extended primer may be melted off by changing thetemperature or pH of the solution, or both the temperature and pH of thesolution in which the sample DNA is solvated. The sample may then bere-sequenced by re-introducing the primer and restoring the temperatureor pH of the solution in which the sample DNA is solvated to theconditions appropriate for primer extension, including appropriateconcentrations of nucleotides and cations.

In some embodiments, the nucleotides may be native dNTPs. In otherembodiments, the dNTPs may be modified, with charge modifyingstructures. The charge modifying structures may be associated, bound orconjugated to the polyphosphate, and subsequently cleaved as part of theincorporation process, obviating the need for a separate process tocleave, separate, or remove the charge modifying structure.

In an alternative embodiment, the charge modifying structure is aterminator and thus be associated, bound or conjugated to the 3′position of the sugar of the dNTP, and may thus act as a terminator.Detection may occur as a result of the process of incorporation, or mayresult from cleavage of the charge modifying structure.

In other embodiments, the charge modifying structure may be associated,bound or conjugated to the 2′ or 4′ positions of the dNTP sugar. In yetfurther embodiments, the charge modifying structure may be associated,bound or conjugated to the base of the nucleotide. The charge modifyingstructures may act as terminators, preventing the incorporation ofadditional dNTPs.

The linkage, association or conjugation may be broken as a result of aphysical process, such as temperature change, or may be broken as aresult of a chemical process, or may be as a result of a photochemicalreaction. The linkage, association or conjugation may be broken aftereach nucleotide incorporation, or several nucleotides may beincorporated, and the number of nucleotides which were incorporated maybe determined as a result of measuring the amount of charge which wasadded as a result of said incorporation(s).

In a further embodiment, two or three nucleotides at a time are used,allowing the addition of multiple bases at a time, and a correspondinglylarge signal. After completing the extension of the primer, withassociated data collection, the extended primer is melted off, newprimer added, and the process of extension may be performed again usinga different order of combinations of dNTPs. This process determineswhich dNTPs do not follow the completion of a previous set of dNTPs,along with information as to the length of the incorporation, whereinsaid length determination need not be exact.

In order to permit repeated measurements of the same DNA sample, the DNAsample may be circularized, whilst the polymerase may be a stranddisplacing polymerase, or may be a polymerase with 5′ to 3′ exonucleaseactivity. Thus the DNA sample may be repeatedly sequenced by allowingthe primer extension reaction to continue for multiple cycles around thecircular DNA sample. In a distinct advantage over a system which usesdetection of fluorophores, the system in certain embodiments of thecurrent invention can employ the full capability of the read length ofthe polymerase, unhindered by having the read length reduced byphototoxicity. In some embodiments, a strand displacing enzyme may beused, thus generating an increase in charge and associated counter ions.In other embodiments a polymerase with 5′ to 3′ exonuclease activity maybe used, allowing net charge to remain the same, while generatingprotons and or hydroxide ions, which may be measured as an increase inconductivity, or may be measured as a result of the ions interactionwith the surface of an ISFET, ChemFET, or NanoBridge sensor.

The polymerase bound or associated with the sensor may be a highlyprocessive polymerase, permitting more bases to be incorporated thenmight occur with a less processive polymerase. The polymerase may bephi29, RepliPHI, MagniPhi®, QualiPhi®, T4 (E. coli T4), F-530, B104, orother highly processive polymerases. The polymerase may be modified, sothat it has reduced or no 3′ to 5′ exonuclease activity, or thepolymerase may have no or little 3′ to 5′ exonuclease activity in itsnative form. Similarly, any 5′ to 3′ exonuclease activity may bemodified so that it is reduced or virtually eliminated. Thermostablepolymerases or other types of DNA or RNA polymerases may be used suchas: Vent (Tli/Thermoccus Literalis), Vent exo−, Deep Vent, Deep Ventexo−, Taq (Thermus aquaticus), Hot Start Taq, Hot Start Ex Taq, HotStart LA Taq, DreamTaq™, TopTaq, RedTaq, Taqurate, NovaTaq™, SuperTaq™,Stoffel Fragment, Discoverase™ dHPLC, 9° Nm, Phusion®, LongAmp Taq,LongAmp Hot Start Taq, OneTaq, Phusion® Hot Start Flex, Crimson Taq,Hemo KlenTaq, KlenTaq, Phire Hot Start I1, DyNAzyme I, DyNAzyme II,M-Mu1V Reverse Transcript, PyroPhage®, Tth (Thermos termophilus HB-8),Tfl, Amlitherm™, Bacillus DNA, DisplaceAce™, Pfu (Pyrococcus furiosus),Pfu Turbot, Pfunds, ReproFast, PyroBest™, VeraSeq, Mako, Manta, Pwo(pyrococcus, woesei), ExactRun, KOD (thermococcus kodakkaraensis), Pfx,ReproHot, Sac (Sulfolobus acidocaldarius), Sso (Sulfolobussolfataricus), Tru (Thermus ruber, Pfx50™ (Thermococcus zilligi),AccuPrime™ GC-Rich (Pyrolobus fumarius), Pyrococcus species GB-D, Tfi(Thermus filiformis), Tfi exo−, ThermalAce™, Tac (Thermoplasmaacidophilum), (Mth (M. thermoautotrophicum), Pab (Pyrococcus abyssi),Pho (Pyrococcus horikosihi, B103 (Picovirinae Bacteriophage B103), Bst(Bacillus stearothermophilus), Bst Large Fragment, Bst 2.0, Bst 2.0WarmStart, Bsu, Therminator™, Therminator™ II, Therminator™ III,Therminator™ Y, T7 DNA, E. coli Polymerase I, Kenow (E. coli) Fragment,Klenow fragment exo−, T4 DNA, Sulfolobus DNA Polymerase IV, AMV ReverseTranscriptase, human polymerase mu, human polymerase mu-h6, DNAPolymerase I (E. coli), T7 RNA (E. coli T7), SP6 (E. coli SP6) RNA, E.coli Poly (A), Poly (U), T3 RNA.

The polymerase and or DNA may be directly bound to or near the sensor,or may be bound through a linker.

In some embodiments, a variant of recombinase polymerase amplificationas described in U.S. Pat. No. 7,270,981, which is hereby incorporated byreference, is used for sequencing. In some embodiments, the amplifiedDNA template may be double stranded, and the input primers may becomplexed with a recombinase such as RecA or RAD51. Said complexedprimers may bind to the double stranded DNA, and with the aid of therecombinase, may displace a portion of the two strands of said doublestranded DNA. A polymerase may then bind to the appropriate end of theprimer such that said polymerase is able to incorporate nucleobases andextend said primer. Single stranded binding proteins may be added, whichbind to the strand of the double stranded DNA which is not hybridized tothe primer. As a result, a larger number of counter ions may be presentfor sensing, whereby, said counter ions may be associated with the newlysynthesized strand of DNA, and additional counter ions may be associatedwith said single stranded binding proteins. An additional advantage isthat issues due to secondary structure resulting from the use of singlestranded DNA template are reduced.

Chamber-Free Reactors and Virtual Reactors

In other aspects, the invention provides a chamber-free device forsequencing a polynucleotide. The device comprises an electromagneticsensor, a magnetic carrier for carrying or holding a templatepolynucleotide to or near the electromagnetic sensor, and a mechanismfor removing the magnetic carrier via liquid flow and/or electromagneticremoval. In certain embodiments, the electromagnetic sensor is one of ananoneedle or a nanobridge, and the device further comprises a localamplifier. The electromagnetic sensor may have a narrow structure, andmaybe etched under the structure such that both sides of the sensor'ssurface are accessible to changes in pH, or to changes in conductivity.

In some embodiments, the system employs magnetic arrays, as described inU.S. Provisional Application 61/389,484 titled “Magnetic Arrays forEmulsion-Free Polynucleotide Amplification and Sequencing, which ishereby incorporated by reference in its entirety. The system is showndiagrammatically in FIG. 5.

FIG. 5 depicts a single element 500 in an array of NanoNeedles andmagnets 516, wherein a substrate 504 may have an electrode 506 on saidsubstrate 504 and under a bead 502, or on a spacing or adhesion layer(not shown) between said electrode 506 and said substrate. Saidelectrode 502 may thus be within the Debye layer of the bead. Adielectric layer 508 may be placed above said substrate 504, and mayalso cover part of said electrode 506, and may further have a recess orcutout which may be larger than the space needed for said bead 502 whensaid bead 502 is retained in place. Said dielectric may serve multiplefunctions, including, providing a surface against which the magneticforce may pull the surface of said bead 502. The magnetic forceresulting from the interaction of magnets 516 and said bead 502, serveto retain and position said bead 502, with a force exerted downwardagainst said electrode 502 and against dielectric 508. A second layer ofdielectric 512 may be applied to said dielectric 508, providingsignificant reagent access to said bead 502, while further extending theheight of the total thickness of dielectric material, such that an upperelectrode 514 may be fabricated on said second layer of dielectric 512.Said upper electrode 514, dielectric, and second dielectric 512, may bepositioned such that said upper electrode may be within the Debye lengthof said bead, and may further be close to the midpoint of said bead 502.Said upper electrode 514 may be above the center line of said bead 502,particularly if said upper electrode 514 is within the Debye length ofsaid bead 502, or may be below the centerline of said bead 502, suchthat the top of said upper electrode 514 is in contact with said bead atan angle of between 10 and 90 degrees from the perpendicular of thepoint of contact between said bead 502 and said electrode 506. Saidangle from the perpendicular to the point of contact between said bead502 and the top of said upper electrode may be between 30 and 85degrees, between 45 and 80, or between 60 and 75 degrees. Said magnets516 may be recessed into said substrate 504, be upon said substrate 504,or upon dielectric 508, but should be below the centerline of said bead502, such that a downward force is applied to said bead 502, such thatsaid bead 502 is pulled down towards said electrode 506. Said magnets516 should further be placed offset with respect to the center of thebead 502 when said bead is in place in the array, such that said bead502 is pulled towards said upper electrode such that said bead 502 isbrought to within a Debye length of said bead 502 to said upperelectrode 514. Each of the elements of single element 500 in an array ofNanoNeedles and magnets 516 may have additional spacer layers oradhesion layers between said elements. Said Debye length may includeDebye lengths which may result from high concentrations of salt, fromlow concentrations of salt, from deionized water, or from aqueoussolutions which are comingled with nonaqueous fluids which are misciblein water.

FIG. 2 is a photomicrograph of a combined virtual well and magneticarray according to various embodiments as described herein. Mostpositions in said array have a single bead in the position between themagnets at the point wherein the virtual well structure is located. Somelocations have more than a single bead. Most of the ends of the magnetsalso have beads located thereupon.

The magnetic array may be used in a manner similar to that described inU.S. Pat. No. 7,682,837, which is hereby incorporated by reference inits entirety.

As used herein, “bead” means beads, moieties or particles that arespherical or non-spherical, where said beads, moieties or particles areporous or solid or a mixture of solid and porous, and can includemagnetic beads that may be may be paramagnetic, super-paramagnetic,diamagnetic, or ferromagnetic.

As used herein, “bead capture features” means features that cantemporarily hold a single bead in a fixed position relative to a sensorand can include local magnetic structures on the substrate, depressionswhich may include an external magnet, local magnetic structures, Van derWaals forces, or gravity as forces that fix the position of a bead. Thebead may be bound in place by covalent or non-covalent binding.

As used herein, “confinement” refers to when a molecule generated (suchas DNA) at one bead or particle stays associated with the same bead orparticle so as to substantially maintain the clonal nature of the beadsor particles.

As used herein “isolate” mean the prevention of migration, diffusion,flow, or other movement, from one virtual well to another virtual wellas necessary to maintain the clonal nature of the beads or particles.

As used herein, “localized magnetic feature” means a magnetic featurecreated on a substantially planar substrate to hold individual beads onsaid substantially planar substrate.

As used herein, “localized magnetic field” means a magnetic field thatsubstantially exists in the volume between the north pole of a firstmagnetic region and the south pole of a second magnetic region orsubstantially exists in the volume between the north and south poles ofa single magnetic region.

As used herein, “particle” means a non-bead moiety such as a molecule,an aggregation of molecules, molecules bound to a solid particle, orparticles, and other forms known in the art.

As used herein, “single phase liquid” is a liquid with relativelyuniform physical properties throughout, including such properties asdensity, index of refraction, specific gravity, and can include aqueous,miscible aqueous and organic mixtures but does not include non miscibleliquids such as oil and water. Among the physical properties notconsidered to potentially cause a liquid to not be considered a singlephase liquid includes local variations in pH, charge density, and ionicconcentration or temperature.

As used herein, “substantially planar” shall allow small pedestals,raised sections, holes, depressions, or asperity which does not exceed40 μm relative to the local plane of the device. Variations due towarpage, twist, cupping or other planar distortions are generally notconsidered to constitute a portion of the permitted offset. Protrusionsor depressions which may be not essential for the uses as describedherein but which exceed 40 μm do not preclude a device from beingconsidered substantially planar. Fluidic channels and or structures togenerate said fluidic channels which have dimensions of greater than 40μm also do not preclude a device from being considered substantiallyplanar.

As used herein, “virtual wells” refer to local electric field or localmagnetic field confinement zones where the species or set of species ofinterest, typically DNA or beads, generally does not migrate intoneighboring “virtual wells” during a period of time necessary for adesired reaction or interaction.

As used herein, “electrode” is defined as any structure used forcreating or applying the electric or magnetic force in such array. Sucha structure may be used for isolation or manipulation in the delivery ofa biomolecule to a special region in the array (e.g. the middle regionof an element in the confinement or isolation array) at a time ofinterest resulting from turning on and off of the fields or forces.

In embodiments of the devices disclosed herein, the device comprises asensing surface for sensing incorporation of a nucleotide, the sensingsurface comprising a layer of silicon nitride.

In embodiments of the devices disclosed herein, a plurality of magneticbeads are configured for carrying template polynucleotide, wherein themagnetic beads have a low zeta potential material at a pH leveleffective for nucleotide incorporation.

The a virtual nanoreactor or “chamber-free array,” may detect ormanipulate particles (e.g., beads, cells, DNA, RNA, proteins, ligands,biomolecules, other particulate moieties, or combinations thereof) in anarray wherein said array captures, holds, confines, isolates or movesthe particles through an electrical, magnetic or electromagnetic forceand may be used for a reaction and or detection of the particles and ora reaction involving said particles. Said “virtual nanoreactor” providesa powerful tool for capturing/holding/manipulating of beads, cells,other biomolecules, or their carriers and may subsequently concentrate,confine, or isolate moieties in different pixels or regions of the arrayfrom other pixels or regions in said array using electrical, magnetic,or electromagnetic force(s). In one embodiment the array is in a fluidicenvironment. Sensing may be done by measurement of charge, pH, current,voltage, heat, optical or other methods.

The chamber-free device described herein in certain embodiments allowsfor better washing of nucleotides during sequencing reactions, mayreduce leading sequencing phase error, by reducing the number ofresidual nucleotides which may include both unbound and nonspecificallybound nucleotides, which may later be inappropriately incorporated in anincorrect cycle.

In addition to DNA sequencing, various different molecular biologyapplications using the “chamber free” array or “virtual” nanoreactor areenvisioned. The array may be used as a tool, for example, as a cellsorter, and subsequently the array may additionally perform molecularbiology on said cells, which may include sorting, measurement ormanipulation for one or more biochemical events or reactions of interest(e.g. drug screening, or biomolecular detection). The virtualnanoreactor array may be used for cell monitoring and analysis, forexample, the system may measure the electrical signature of a cell wheresaid cell is captured in the array and or adjacent to sensing elementsassociated with the virtual nanoreactor. The array may be used forscreening of rare cells, for example, for detection of reactions in drugscreening for drug development, or for selection and testing ofcancerous cells. In various embodiments assays or detection targetsinclude cell biology, drug screening and monitoring for specific celltypes, detection of DNA, RNA (nucleic acids), proteins, charged smallmolecules, ligands, or other biomolecules.

In other embodiments, a further electrode (or virtual wall/fence)element may enclose the other two electrodes associated with eachelectrical confinement or isolation element in the array.

In some embodiments, the system is used to capture multiple beads orcells per pixel, and may turn on, turn off or modify the magnitude,shape or period of the electromagnetic field at a desired time or inresponse to a change at a sensor associated with said element in thearray of “virtual walls” as may be needed for different applications.For example the system may capture one set of beads or cells and thenincrease the field strength to capture another moiety which is lessinfluenced by said field. The electric field can be a DC or AC orcombination of different combinations of the fields.

FIG. 3 is a graphical representation resulting from a Comsol simulation,depicting the 3D equipotential field strength curves 306, of one fourthof a cylindrical structure 300 that result from a field being applied tothe electrodes 304A and 304B. As a result of the substantial radialasymmetry of the field, the volume where the field gradient is mostconcentrated 308 near the center electrode 304 B, which is the pointwhere a bead may be held by a magnetic array as shown in FIG. 2.

In other embodiments, the virtual nanoreactor(s) are used for and orcombined with multiple steps in a biomolecular process, for example,bead enrichment of particle(s) moving in an electric field, microfluidicsample-prep and library prep such as on-chip extraction of DNA, shearingof DNA, on-chip normalization of DNA concentration, emulsion-freeamplification, sensing, which may include SensePlus sensing utilizingdual-sensing or multi-sensing sequencing detectors, which may utilizetransient and or steady state electronic sequencing and rephasingmethods for extending the length of read of a sequencing process,wherein said multiple steps may result in a fully integrated electronicgenomic analyzer system.

In some embodiments, multiple arrays of virtual nanoreactors are used.The different arrays of virtual nanoreactors may be used for differentbiological reactions, processes or methods. In some embodiments, onearray or set of arrays is used for extraction of DNA, where differentarrays in said set of arrays or different members within a set mayretain cells from different samples, or may retain different types ofcells from a single sample, or a combination thereof. For example,different samples may be held in different arrays, and different typesof cancer cells from a single sample tumor may be retained withindifferent areas of a single array. The different cell types may besorted or segregated, or the cell type may be determined as a result ofdata derived from the individual cells after said cells are captured andretained in individual locations on said array of virtual nanoreactor.

In some embodiments, several biological reactions, processes or methodsmay be performed on a single sample or moiety while it is retained inposition within a virtual nanoreactor. In other embodiments, one or morebiological reactions, processes or methods may be performed on asample(s) or moiety(s) in a position(s) in a virtual nanoreactorarray(s) prior to the transfer or movement of said sample(s) ormoiety(s) to another position(s) in another virtual nanoreactorarray(s).

In some embodiments, the array of electrical concentration andconfinement virtual nanoreactors is used for purposes other than thecapture/isolation of nucleic acids as otherwise described herein forsequencing and amplification of nucleic acids. In some embodiments, theelectrical concentration and confinement is used with any chargedmoiety, where the time period, moiety concentration, mobility,concentration, local viscosity, cross-linking percentage of localpolymers, or concentration of the charged moiety(s) influences thespacing of electrical confinement structures, and or field strengthused, and or magnitude of field gradient in order to maintain sufficientconfinement within individual virtual nanoreactors. The level of crosscontamination between different virtual nanoreactors which may betolerated may vary for different applications, and at different steps ina biological reaction, process or method. For example, if a nucleic acidamplification reaction process was performed on different samples inadjacent virtual nanoreactors, cross contamination may be problematicduring early thermal cycles of a PCR reaction, while cross contaminationduring later cycles of a PCR reaction may not be particular concern, asprimers may be largely consumed, preventing significant amplification ofthe cross contaminating nucleotides from other virtual nanoreactors.

In some embodiments, a polymer is used to reduce the migration rate ofnucleotides or other moieties. For example, the polymer may be apartially entangled polymer such as POP-7™, or an agarose,polyacrylamide, or starch gel. The concentration and or percentage ofcross linking may be varied as needed for various mobilities ofdifferent moieties for which confinement in the virtual nanoreactor isdesired. The polymer may be introduced to the volume of the virtualnanoreactor before, with, or after the introduction of sample moietiesor other moieties used in said biological reaction, process, or method.

In some embodiments, the electrical concentration and confinement isused to capture moieties to which other biomolecules or biologicalmoieties have been bound, for example beads with charge to whichantibodies may be bound, and wherein said beads may be subsequently usedto capture proteins, and wherein said beads further may be subsequentlycaptured using the electrical concentration and confinement array.

In some embodiments, the virtual nanoreactors may be used for severaldifferent applications in addition to sequencing, for example saidvirtual nanoreactors may be utilized as a hybridization array (similarto an Affy or Agilent DNA micro array) wherein the DNA is on beads, andsaid beads are held in place by the virtual nanoreactor. In otherembodiments, the virtual nonreactors are used for digital PCR, wheresaid beads are introduced into the array. Detection may be done with anelectronic sensor array associated with each element of the array, ordetection may employ optical means, to detect the presence or quantityof a particular nucleic acid. Digital PCR may be used to quantify theconcentration of targets within a sample in a relativistic manner.

In some embodiments, concentration of sample into the virtualnanoreactors as described herein allows quantitation with greatersensitivity. In some embodiments, using concentration in some areas ofthe array and not in others may allow for a greater concentrationquantitation dynamic range. In further embodiments, DC concentrationfields may be inverted so as to partly “push” sample moieties away fromsaid virtual nanoreactors. In some embodiments, combinations ofconcentration, no concentration, and pushing away of sample moieties todifferent areas may help to further extend the dynamic range of digitalPCR, or of any other desired biological reaction, process, or method.Beads with different primers may be introduced into different areas ofthe virtual nanoreactor array as described herein, allowing simultaneousdigital PCR reactions for several targets.

In some embodiments, the virtual nanowells are used for full extensionreactions of DNA bound to primers conjugated to said bead. In a furtherembodiment, said virtual nanoreactor may be used for ligation reactiondetection.

In some embodiments, said system may use electromagnets, permanentmagnets, or electrodes or other different subsystems to generateelectromagnetic fields for transient or part-time isolation, or holdingor concentration of the biomolecules or other moieties of interest (e.g.DNA, cells, proteins) or the carrier of the biomolecule or other moietyof interest (e.g. beads, particles, or other moieties). Saidelectromagnetic fields may be magnetic fields, electric fields, or acombination of the two. Said electric fields may be DC fields, ACfields, pulsed DC fields, non-sinusoidal AC fields, pulsed AC fields, ora combination thereof. The nanoreactors with virtual walls (or fences)may be used or created with electric or magnetic fields to hold,capture, concentrate, isolate, or manipulate the biomolecules or itscarrier.

In some embodiments, where two or more sets of electrode structures areemployed, virtual walls or isolation walls or fences may be turned on oroff, or modified as to the magnitude, shape or period of theelectromagnetic field at a desired time, which may be a fixed time, ormay be in response to a change from a sensor associated with a specificmember of the array of electrode structures. Turning on or off, ormodifying as to the magnitude, shape or period of the electromagneticfield may be used for controlling the movement of the particles, beads,cells, biomolecules or other moieties of interest which areconcentrated, confined, or isolated in the array of electrodestructures, or for controlling the biomolecules or other moieties ofinterest which may be used in a reaction, such as an antigen orsecondary antibody in protein detection, or nucleotides in DNA or RNAsequencing, or secondary cell in cellular interactions, or drugs orcells in drug screening and monitoring. This feature may be used toprovide easy access and flexible manipulation, and or mixing.

In some embodiments, the virtual nanoreactor array normalizes the amountand/or concentration of DNA input into the system, and may generate afeedback and/or control to the entry, which may then be used to controlthe amount and/or the concentration of said DNA, or other biomoleculesor other moieties input into the system as described herein. The systemmay be further used for the purpose of real-time normalization of thedetection or sequencing array.

The array for capturing (isolating, confining or concentrating) ofbeads, or cells, or other biomolecules, particles, or other moieties ofinterest through magnetic or electromagnetic or electric capturing andor holding may be structured such that it comprises two sets of the“capturing elements” (GENIUS bars or elements of a magnetic array asdescribed elsewhere) per element of the array, allowing the capture(isolating, confining or concentrating) of one moiety with one set ofcapturing elements, and the subsequent capture (isolating, confining orconcentrating) of a second moiety of interest. Said capturing can bedone in different orders and with different structures, and may includemore than two sets of capturing elements per array element, andcorrespondingly, additional capture steps may be performed at each arrayelement.

In some embodiments, the electrical concentration and confinement arraymay be used to capture charged beads to which B-cells have been bound,for example magnetic beads to which proteins, polysaccharides, or otherimmunogens are carried, and where the beads may be subsequently used tocapture B-cells. The charged beads may be subsequently captured usingsaid electrical concentration and confinement array. In someembodiments, the electrical concentration and confinement array is usedto capture B-cells to which proteins, polysaccharides, or otherimmunogens may be bound.

In some embodiments, the electrical concentration and confinement arrayis used to capture charged beads to which other carbohydrates orglycolipids have been bound, for example charged beads to which proteinsor peptides which comprise carbohydrate binding modules may be bound.The charged beads may be subsequently used to capture carbohydrates orglycolipids, and where the charged beads further may be subsequentlycaptured using said electrical concentration and confinement array. Sucha carbohydrate or glycolipid binding moiety may comprise a carbohydrateactive enzyme, such as a glycosidic hydrolase, a lectin, a galectin, aintelectin, a pentraxin, a selectin, an adhesion, or a hyaluronan.

In some embodiments, the array is used for chemical screeningapplications and non-biomolecules can be tested or monitored or measuredwith the system. For example, in use for drug screening, wheremeasurement of a drug effect is desired, the system may employ, forexample, about 100, 1000, 10,000, or 1,000,000 different drug candidateseach on their own beads, where said beads may be captured and hold inthe “virtual nanoreactor” array. The system may subsequently allowinteractions and measurements of the interactions between the beads anda cell or set of cells of interest, where the electrical confinement isused for isolation of the pixels in the array, providing a highthroughput and fast drug screening system.

In some embodiments, the electrical concentration and confinement arrayis used to capture a combined set of charged beads to which multipledifferent types of biomolecules have been bound. The combined set ofcharged beads may comprise multiple sets of charged beads with differenttypes of biomolecules or biological moieties bound. The may be one typeof binding moiety which may bind one biomolecule or biological moiety ona set of charged beads, and a different type of binding moiety which maybind a different biomolecules or biological moiety on a different set ofbeads. Said combined set of charged beads may also comprise sets ofcharged beads wherein the set of charged beads may comprise chargedbeads to which multiple binding moieties are bound.

In further embodiments, said combined sets of beads may further comprisea label, wherein the label differentiates between different sets ofbeads. Said label may be an optical label, such as a fluorescent dye, abiochemical label such as DNA, metal particle labels, any other type oflabel, or a combination of different types of labels. Said labels may beused to determine which type of bead may be at each element of the arrayelectrical concentration and confinement features.

In some embodiments, different methods of detection of the beads and theinteractions that result from various biomolecular reactions may beobserved as a result of the detection of said beads. Said detection maybe effectualized as a result of a NanoNeedle sensor, a NanoBridgesensor, a ChemFET sensor, an ISFET detector, an optical sensor, such asfor example a fluorescence detector, a SERS detector, an absorptiondetector, a PH detector, a conductance detector, a mass resonancedetector, a calorimeter detector, or any other type of detector suitablefor detection of biomolecular reactions, or of other types of reactions.In some embodiments, said sensors may be combined at each position inthe array with an electrical concentration and confinement feature.

In some embodiments, the electrical concentration and confinement areused to directly capture charged moieties, where said charged moietiesare bound to other biomolecules or biological moieties. For exampleantibodies or antibodies bound to proteins may be concentrated orconfined, where said antibodies or antibodies bound to proteins may besubsequently used to capture proteins or additional antibodies.

In some embodiments, the “virtual nanoreactor” array increases thereaction rate by concentrating biomolecules of interest, such as DNA,RNA, or other reactants or reagents for more efficient synthesis.

In some embodiments, it is desirable to integrate a valving system aspart of a flow cell. Said valving system enables the flow of samples tosections of a flow cell, such that different samples may be used fordifferent sections of said flow cell. In other embodiments, the valvingsystem is integrated adjacent to the flow cell, whereby the valvingsystem and flow cell may form a sealing interface to each other. Inother embodiments, said valving system and said flow cell may beadjacent to each other on a single mount, wherein both said valvingsystem and said flow cell may be mounted to said mount. Said valvingsystem may also comprise a waste valve(s) such that fluids may beremoved from said valving system prior to flowing into said sections ofsaid flow cell. For example if there is a significant dead volume insaid valving system, it may be desirable to remove fluid which may havean unacceptable level of cross contamination from a previous fluid.

In some embodiments, it may be desirable to integrate a valving devicewith the flow cell. Such a valving configuration may include variousinputs, which may include inputs for the four dNTPs (e.g., forsequencing reactions), which may also contain buffer, salts, enzyme andany other moieties required for incorporation of nucleotides. Inputs mayalso be employed for various buffers and wash reagents, polymerasecontaining buffers, which may also contain salts and any other moietiesneeded for polymerization, reagents needed to strip any coatings fromthe flow cell, reagents which may be needed to re-coat the flow cell,buffers which also include a phosphatase, or other reagents.

In one embodiment, the valving device is fabricated of PDMS. In anotherembodiment the valving device is fabricated from glass with magneticallyor pneumatically activated elastomeric valves.

In some embodiments, it may be desirable to bond said valving andfluidics PDMS manifold to a silicon device. It may be desirable toincrease the bonding strength between said PDMS and said silicon device.

In one embodiment of the current invention, it may be desirable to useplasma activated PDMS to improve the bond strength. As a plasmatreatment which has too much power or too much pressure may actuallydecrease the bond strength of PDMS to silicon, it may be important touse lower power levels and pressures. Tang et al describe appropriatepower and pressure levels in 2006 J. Phys.: Conf. Ser. 34 155. In oneembodiment it is suitable to use a pressure between 500 mili Torr and 30miliTorr and a power level between 10 and 60 watts while using, forexample, a 790 series Plasma-Therm.

For a device fabricated of PDMS or other similar materials, it ispossible to use pressure valves to control the flow of reagents. Withsuch valves it is possible to have the several valves in close proximityto each other, and the valves may be very close to a central channel,reducing dead volume, as shown in FIG. 4A, which shows a reagent valvesystem 400 with three reagent input lines 402 with valves 406, each ofwhich can be configured to flow towards the input to the flow cell 408,under the control of pressure control lines 404.

For a more complex system, where more reagent inputs are desired, thesimple valve system 400 of FIG. 4A is insufficient, as it has but threereagent inputs lines 402. In an alternative embodiment as shown in FIGS.4B and 4C, many more inputs are enabled. This approach also permitsclearing the dead volume of the channel. In FIG. 4B, inputs includeinput ports for dATP, dTTP, dCTP, dGTP, Buffer one, Buffer two, andSample, output ports Waste one, Waste two, and Waste three. Controllines are in place for each input and output port, with additionalcontrol lines to control the direction of flow between activated ports.A waste port is shown immediately prior to the flow cell, so that anyremnant reagent from a previous flow may be removed, allowing a veryclean transition from one reagent to another, without diffusion from anydead volumes in the valving system. FIG. 4C depicts a valving systemwith a oval flow path, such that all input valve port positions have apath to an outlet (waste) port in both directions from said input valveport position. Valves as shown in FIG. 4A may be used for each valveshown in FIGS. 4B, 4C or in a physical embodiment of a reagent valvingsystem 430 as shown in FIG. 4D, wherein photograph of a PDMS valvesystem 440 is shown.

One embodiment permits purging of air or other contaminants in eachinput line up to each input port control valve, so that when each inputport is activated, appropriate reagents may be introduced to the system.For example, to clear the dATP line, the dATP control line and the Wasteone Upper Control line may be activated causing air and any unwantedreagents in the dATP lines to flow through the dATP valve and out Wasteone. In other embodiments, the dCTP, dTTP, and the dGTP lines may bepurged or cleared of contaminants by activating the dCTP control lineand the Waste one Upper Control lines, the dTTP and the Waste one UpperControl lines, and the dGTP control lines and the Waste one LowerControl line respectively. To purge or remove contaminants from thesample line, the Sample Control line and the Waste two Control lines maybe activated.

Purging and removing contaminants from all lines may be needed afterreplacing a sequencing assembly. Similarly purging or removingcontaminants from reagent lines may be needed after replacing orrefilling reagent bottles or containers. A further need for purging orremoving of contaminants may result from periods of time wherein theinstrument is not used, which may permit any reagent lines containingreagents which need to be cooled below ambient to suffer degradation;for example, polymerase in a polymerase containing reagent may sufferfrom extended exposure to ambient temperatures.

In some embodiments, it is desirable to fill the manifold leading to theinput of the flow cell, so that any reagents remaining from a previoususe of the manifold may be removed. For example, prior to introductionof dATP into the flow cell, the dATP control line, the Upper LiquidControl line, and the Waste two Control line may be activated. dATPreagent will then commence to flow from the dATP input line, around bothsides of the upper liquid loop, through the channel between the upperliquid region and the lower liquid region, and out through the waste twovalve into the waste two line. Alternatively, prior to introduction ofdCTP into the flow cell, the dCTP control line, the Upper Liquid Controlline, and the Waste two Control line may be activated. dCTP reagent willthen commence to flow from the dCTP input line, around both sides of theupper liquid loop, through the channel between the upper liquid regionand the lower liquid region, and out through the waste two valve intothe waste two line. Similarly, prior to introduction of dTTP into theflow cell, the dTTP control line, the Lower Liquid Control line, and theWaste two Control line may be activated. dTTP reagent will then commenceto flow from the dTTP input line, around both sides of the lower liquidloop, through the channel between the lower liquid region and the lowerliquid region, and out through the waste two valve into the waste twoline. Similarly, prior to introduction of dGTP into the flow cell, thedGTP control line, the Lower Liquid Control line, and the Waste twoControl line may be activated. dGTP reagent will then commence to flowfrom the dGTP input line, around both sides of the lower liquid loop,through the channel between the lower liquid region and the lower liquidregion, and out through the waste two valve into the waste two line.

Buffer one can be made to flow through the main flow cell (in darkblue), and out the Waste three port by activating the B1C control line,and the W3C control line. Alternatively, Buffer one can be made to flowout the Waste two port by activating the B1C control line, and the W2Ccontrol line. In another alternative use, Buffer one can be made to flowout the Waste one line through the upper section of the liquid manifoldby activating the Buffer one control line, the Upper Liquid Controlline, and the Waste one Upper Control line. Similarly, the lower liquidmanifold can be flushed with Buffer one by activating the Buffer onecontrol line, the Lower Liquid Control line, and the Waste one LowerControl line. Activating flow in a combination of these areas either ina time sequence or activated together enables clearing the entire liquidmanifold to be purged of bubbles, other contaminants, or as a wash orpurge of any other liquids which may have been introduced to the systemvia the dATP, dTTP, dCTP, dGTP, Buffer two, or Sample input ports.

In some embodiments, it is desirable to use a passivation layer over thesilicon device which has a higher bond strength than thermally grownsilicon dioxide. Tang et al describe several passivation layers whichprovide improved bonding strength, including PSG (PECVD phosphosilicateglass), USG (PECVD undoped silicate glass), Si₃N₄(LPCVD siliconnitride).

In some systems which use PDMS valving manifolds, pins or needlesinserted into the PDMS are used to connect reagent lines to the valvingmanifold. While this provides for secure attachment, attaching a numberof reagent lines to a PDMS valving manifold is time consuming and errorprone. Thus in some embodiments, it may be desirable to use an interfacemanifold, where the reagent lines are connected to the interfacemanifold, rather than to the valving manifold, and the interfacemanifold may be connected to the valving manifold. The reagent lines maybe attached to pins or needles, which may be attached to the interfacemanifold. The pins or needles may be permanently affixed to theinterface manifold, being held in place with an adhesive, by welding orbrazing, by utilization of a press fit, or by some other means.Alternatively, the lines may be directly connected to the interfacemanifold, where they may be retained by a fitting or o-ring, or by someother means as known in the art.

In some embodiments, the interface manifold may sealingly interface tothe valving manifold such that reagents may flow from the interfaceblock to the valving manifold. The interface between the interfacemanifold and the valving manifold may be an interface which is used by auser to enable replacement of the chip/flow cell and/or valvingmanifold.

In some embodiments the interface manifold may have internal channelsformed by bonding, such bonding could include fusion bonding, solventbonding or adhesive bonding.

In some embodiments, minimizing the path length to the active part ofthe flow cell may be important for several reasons, including minimizingthe amount of mixing of reagents, which occurs as a result ofdifferences in flow rates at the center of a channel versus the flowrate at the edges of a channel, due to wall interactions, as well asdiffusion. In some embodiments it may also be desirable to minimize thevolume which is not temperature controlled, in order to preventdegradation of reagents, such as polymerase, in volumes which are nottemperature controlled. In some embodiments it may be desirable tominimize plumbing volume to concomitantly minimize cross contaminationof reagents may also occur in regions of flow which are common tomultiple reagents, due to nonspecific binding to materials which contactsaid reagents.

FIG. 1 illustrates a schematic representation of one embodiment of thecurrent invention, where a magnetic or paramagnetic bead is held inplace over a sensing region by a magnetic array. The magnetic array isdescribed in U.S. Provisional Application 61/389,484 titled “MagneticArrays for Emulsion-Free Polynucleotide Amplification and Sequencing,”which is hereby incorporated by reference in its entirety. Retainedmagnetic or paramagnetic beads may have monoclonal populations of DNA.The beads may be sized such that there is sufficient room for only onebead over each sensor, thus providing for a one to one correspondencebetween sensors and beads. Although there may be room for only one beadover each sensor, there can be room between beads when the beads arealigned over the beads, resulting in reduced cross-talk between sensors.For example, a set of beads may be about 10 microns in diameter locatedover sensors which are about 8 microns across, and the sensors may bespaced about 15 microns apart, resulting in an approximately 5 micronspace between the beads. The size of the sensors may be larger than thebeads, if there is insufficient room for two beads to be retained abovethe sensor. The size of the beads, sensors, and spacing can vary. Inother embodiments, beads may be greater in size than 10 microns, such asabout 15 microns, about 20 microns, about 25 microns, or larger. Infurther embodiments the beads may be smaller than 10 microns, such asabout 5 microns, about 3 microns, about 2 microns, about 1 micron, orless than one micron. The sensors may be sized to align with the size ofthe beads, and thus may be larger, or smaller in size than 8 micronsacross, potentially ranging from less than one micron, to about 1, 2, 3,5, 10, 15, 20 or more microns across. The spacing between the sensorsmay also be greater than 15 microns, or may be less than 15 microns; thesensor spacing may range from less than one micron, to about 1, 2, 3, 5,10, 15, 20, 25 or more microns between sensors.

A chamber-free magnetic retention structure as shown in FIG. 1 maypermit improved flow of nucleotides, polymerase and other components, astheir flow is not hindered by a well structure, such as that shown inFIG. 2, permitting better washing, more complete incorporation of bases,and faster cycle times then would be possible if the bead were in awell. In a well structure, a bead and associated DNA such as shown inFIG. 2 hinders accessibility and flow, so that a higher concentration ofpolymerase and nucleotides may be needed to permit sufficient diffusionto all parts of a bead as shown in FIG. 2. Said higher concentrations ofdNTPs and polymerase may increase the error rate due to misincorporationby said polymerase, resulting in higher levels of leading sequencingphase error then might occur with a chamber-free structure such as isshown in FIG. 1.

In some embodiments, the array described herein is reusable (e.g., notsingle use). The cost of sequencing has a number of parts; forsequencing using electronic sensors, one of the major costs is the costof the processed silicon itself; that is: the sensor. This may beparticularly true if the sensor is not re-useable, but must be discardedafter a single use. The magnetic array described above makes reusefairly straightforward, as the DNA is not bound to the sensor, and thebeads can be easily removed by reducing or removing the magnetic fieldwhich holds said beads in place. If the beads are instead held in placewith a structure, removal may be more difficult.

In one embodiment, beads which are held in place by a structure, orarray of wells, and removed by applying a magnetic field such that thebeads, which may be magnetic or paramagnetic beads, are pulled out ofthe wells, and subsequently removed from the flow cell by flowing areagent through the cell.

In some embodiments, the array of magnetic features are used forpurposes other than the capture/isolation of nucleic acids as otherwisedescribed herein for sequencing and amplification of said nucleic acids.These include capture of cells (e.g., cancer cells, B-cells), proteins,glycoproteins, glycolipids, antibodies, saccharide or polysaccharide,and other moieties as already described.

In some embodiments, associated cells bound to retained beads are lysedwhile the beads are held in position in the magnetic array. The retainedbeads may further comprise attached primers or primer sets foramplification and/or sequencing target nucleotide sequences. The primersmay be universal primers, or primers targeted to specific sequencesassociated with the type of cell bound to each bead, or may be universalprimers or universal primer sets with barcodes. For example, barcodesare associated with the cell type bound to each bead, or a combinationof different primer types. In some embodiments, after lysing of cells, areverse transcription and/or amplification reaction may be performed. Insome embodiments, the amplification is a real time PCR reaction, wherethe quantity of a specific RNA may be determined for each bead and thuseach cell type. In other embodiments, the amplification reaction is aPCR reaction or isothermal reaction, and may generate clonal populationson said beads, or may generate multiclonal populations, where each clonetype may use different primers. In other embodiments, a sequencing bysynthesis reaction is subsequently performed to determine the sequenceof the amplified sequence(s) associated with each bead type, and thuswith each cell type.

In further embodiments, after lysis of cells, DNA, RNA or othermolecules of a specific charge may be retained, and the beads may beremoved. In some embodiments, additional beads may be introduced to saidmagnetic array. The newly introduced beads may have different primertypes associated with the newly introduced beads as described herein.

In some embodiments, the array of magnetic features may be configuredsuch that a preferred position is maintained by a magnetic orparamagnetic bead or particle. Such a preferred position may be desiredso as to appropriately position the particle with respect to a sensor orsensors, and/or to maintain the particle in a fixed location, so thatthe particle and the charge attached to the particle does not move withrespect to said sensor or sensors. In some embodiments the preferredlocation may result, at least in part from the configuration of themagnetic array element shapes, as shown FIG. 6. FIG. 6 illustrates twodifferent configurations 600 wherein at least some of the shapes in thearray may be configured such that the density of the magnetic flux ismore concentrated at one end of members of the magnetic array than atthe other end or some other members of the magnetic array. In the toptwo pairs of magnetic elements, the left trapezoidal magnetic arrayelement 604 is narrower at the south pole, than at the north pole ofsaid trapezoidal magnetic array element. As the total flux levelemanating from the magnetic array element must be the same at the twoends, the flux density at the narrower end of the trapezoidal magneticarray element 604 will be higher than at the wider end of saidtrapezoidal magnetic array element. As a higher concentration of fluxcorresponds to a higher force exerted on a magnetic or paramagneticelement, a higher force will be exerted on the magnetic or paramagnetparticles or beads 602 by the narrower end of said trapezoidal magneticarray element 604, than is exerted by either the similarly sizedmagnetic array element 606 on the right side wherein the element on themagnetic array element on the right side 606 is of similar size, but isrectangular, and thus has a lower flux density and force. Similarly, ahigher force will be exerted on the magnetic or paramagnet particles orbeads 602 by the narrower end of the trapezoidal magnetic array element604, than is exerted by either the similarly sized trapezoidal magneticarray element 608 on the right side wherein the element on the magneticarray element on the right side 608 has its wider end oriented towardsthe magnetic or paramagnetic particle or bead 602.

In another embodiment of the current invention, additional features maybe incorporated as part of fabrication of the magnetic array so as toinhibit motion of the magnetic or paramagnetic particle, or bead 702 asshown in FIG. 7. In the embodiment shown, the magnetic or paramagneticparticle or bead 702 is pulled preferentially towards the narrow end ofthe left trapezoidal magnetic array element 704, and is pulled intocontact with two posts 710, as well as being pulled down into contactwith the surface of the array, thus providing three points of contact tofully stabilize said magnetic or paramagnetic particle or bead 702. Theposts and magnetic array elements may provide minimal areas of surfacecontact, so as to permit maximal access by ions, dNTPs enzymes and othermoieties. The posts and magnetic array members may be positioned withtight tolerances with respect to the sensor elements, so as to providereproducible signal levels between different members of the sensorarray.

In further embodiments, a small well is used, such that a magnetic orparamagnetic particle may rest on the upper corners of the well. Thewell may be round, or may be of some shape other than round if themagnetic or paramagnetic structure is generally spherical in shape, soas to allow better access to the bottom of the magnetic or paramagneticparticle by enzymes, dNTPs, ions and other moieties.

In some embodiments, the magnetic array is used to generate clonalpopulations for hybridization detection, hybridization pullout, orsequencing. The assay may be performed with beads in the positions inthe magnetic array where the amplification occurred, or the beads may bemoved from the area or volume where the amplification reaction tookplace to another location. The second location may also employ amagnetic immobilization to perform the assay, or may employ a differentimmobilization such as biotin streptavidin binding. In some embodiments,the sensors are positioned directly under the magnetic or paramagneticparticle, so as to maximize the interaction between the chargeassociated with the DNA on the bead and the sensor. In otherembodiments, the bead may be bound or associated in such a manner thatit is fixed and unable to rotate freely. It may be desirable to positionthe sensor off center from the paramagnetic, permitting access to areasof the particle which have free access to the aqueous environment andaccess to polymerase, dNTPs and other moieties is permitted and may haveoptimal enzymatic reaction which may then be read by the sensor, incontrast to the areas which are in direct contact with the surface wherethe reaction may be inhibited as a result of lack of access to theaqueous environment.

In some embodiments, it is desirable to use the differential flow whichoccurs in a channel to rotate the magnetic or paramagnetic particles, soas to provide optimal access by enzymes, dNTPs and ions to all surfacesof said magnetic or paramagnetic particles. Said differential flowresults from the parabolic flow typical of small channels, where thebulk flow rate at the surface of a channel is zero, and the maximal flowrate in the channel is typically highest in the center of said channel.This may result in a significant flow rate differential between thebottom of the channel and the top of the magnetic or paramagneticparticle. The difference in flow rate between the top and bottom of themagnetic or paramagnetic particle will be a function of the size of theparticle, the height and width of the channel, and the average flow ratein the channel. The DNA and or other moieties which may be attached tothe magnetic or paramagnetic particle may provide a drag or pull on thetop of the magnetic or paramagnetic particle due to the comparativelyhigh flow rate at the top of said magnetic or paramagnetic particle. Theflow rate at the bottom conversely will remain essentially zero, thuscreating a significant rotational impetus. The magnetic array elements,potentially combined with other physical features may maintain theposition of said magnetic or paramagnetic particle.

In some embodiments, the flow rate is maintained at a consistent flowrate while introducing and flowing dNTPs and or other reagents whilereading the sensors, thus maintaining a consistent average rotationrate. In other embodiments, it is desirable to reduce the flow ratewhile reading the sensor, so as to prevent significant oscillations andmotions of the magnetic or paramagnetic particle. In yet otherembodiments, it is desirable to increase the flow rate while flowingreagents through the flow cell when reading the sensor, so as to permitmore surface area of the magnetic or paramagnetic particle to interactwith the sensor. This may permit an increased averaging effect, whichmay reduce sensor readout variations due to variations in DNA attachmentdensity on the surface of the magnetic or paramagnetic particle, orvariations in the sensor readout due to irregularities in the shape ofthe particle.

In other embodiments, forces other than that resulting from changes inthe flow rate velocity are used to modify the rate of rotation ormovement of the magnetic or paramagnetic particle. In some embodiments,the flux levels may be modified as a result of the movement of anexternal magnet which may be coupled through the magnetic array elementsas a result of the higher permeability of the magnetic array elements.In an alternative embodiment, and electromagnet is used to influence theamount of flux which interacts with the particles. These changes in theamount of magnetic flux may reduce the frictional forces acting on themagnetic or paramagnetic particle, permitting more or less rotation.

In yet other embodiments, a magnetic or electric field is used to rotatethe magnetic or paramagnetic particles, in part as a result of thepolarizability of the bead and associated DNA.

In some embodiments, it is desirable to use a force in addition to thatwhich results from the flow of reagents through the flow cell toposition magnetic or paramagnetic particles into appropriate locationsassociated in a one to one correspondence with sensors. In someembodiments, magnetic or paramagnetic particles may be flowed in areagent stream into a flow cell or cells, and a magnet or electromagnetis used to move the particles into positions associated with sensorssuch that a higher proportion of the magnetic or paramagnetic particlesis associated in a one to one correspondence with sensors than wouldhave occurred without the use of such additional magnets orelectromagnets.

The magnetic array also permits virtually complete allocation of beadsto array locations. Low speed flow is sufficient to enable localizedretention of the beads in the array, in a one to one correspondencebetween beads and array locations, without requiring centrifugation. Inone embodiment, if even higher levels of filled versus unfilledlocations on the array is needed or desired, the reagent flow may becircularized, such that the beads may be reintroduced to the flow cell.In another embodiment, the reagent flow may be stopped or slowed as thebeads may be introduced to the flow cell. In another embodiment, thedirection of the reagent flow may be reversed, potentially severaltimes, providing more opportunities for the beads to fill the array. Inyet another embodiment, the beads are retained after flowing the beadsinto a flow cell by flowing the beads through either the inlet or outletto a storage location, so that they can be used in a subsequentsequencing process. To prevent beads from sticking in positions otherthan the intended bead locations, the flow can be increased to removeany weakly held beads, yet retain the correctly held beads. After achemical process such as sequencing or amplification has been completed,the beads may be removed by either reducing the retention field flux, byadding a new field that pulls the beads away from the array, byincreasing the flow rate of the fluid, by using the air water interfaceassociated with an air bubble, which may include a surface tensionforce, or any combination of the above steps.

In an certain embodiments, as shown in FIG. 3, an array of electrodes isused to retain charged beads, using either a DC field or adielectrophoretic field, or both. As with the magnetic array, no wellstructure is needed to retain the bead, permitting free flow ofcomponents in solution. To insure that charged components in solution,such as the DNA sample, nucleotides, enzymes and other charged moietiesmay be readily able to flow through the volume above the array, afrequency of oscillation which is sufficient to retain the charged beadsis used, but which is sufficiently slow as to permit the moieties insolution to flow or diffuse away from a bead which is retained. Theaddition of depressions associated with the sensors in a one to onecorrespondence may result in better alignment between the beads and thesensors permitting better detection. In an alternative embodiment,pedestals or registration posts or other three dimensional structuresare used, for example, for better fluid flow may be incorporated.

In an alternative embodiment, wherein the beads may be localized in aone to one correspondence to the array, the beads may be brought intoposition by a magnetic or electrical field, and may then be held inplace by an alternative means, such as DNA hybridization, biotinstreptavidin binding, thiol binding, photo-activated binding, covalentbinding, or the like. The binding may be initiated by a change intemperature, application of light, or by washing in a binding reagent orcatalyst whilst the beads are held in a one to one correspondence bysaid magnetic or electric field. After binding has occurred, themagnetic or electric field strength may be allowed to change inintensity or frequency, potentially being turned off. The binding may bereversible, permitting the beads to be washed out of the volume abovethe array of sensors.

In some embodiments, the magnetic or paramagnetic particle may havesurface coating thereon that is of sufficient porosity to provide accessfor a polymerase or other enzyme, as well as for sample DNA, dNTPs ionsand other moieties to pass through. The coating may be configured sothat primers may be attached at an appropriate spacing, and may therebyprovide a greater density of sample DNA and thus charge to interact withthe sensors located thereby. Said coating may be of agarose,polyacrylamide or other cross linked polymer, or may be made of porousglass.

In other embodiments, the bead may have coatings configured so as tominimize or reduce nonspecific binding of DNA, proteins, or othercharged moieties relative to the amount of nonspecific binding which mayresult when said DNA proteins, or other charged moieties interact withsaid beads without said surface coating. Said coatings may be similar tocoatings described herein for use on the surface of a flow cell, sensor,enrichment module, or magnetic array, and any coating herein describedfor one surface may be utilized on other surfaces.

In some embodiments, the beads have a magnetic core, which may have animpermeable coating thereupon. Said coating may be bound, attached orassociated with multiple strands of DNA. For example, the DNA strandsmay each be substantially identical rolling circle amplicons, providingmultiple strands of DNA where each strand has multiple contiguous copiesof one DNA target.

FIG. 8 illustrates one embodiment for an alternative method and systemfor retaining beads in a one to one correspondence with the sensors inan array. FIG. 8 illustrates a system 800 whereby individual controllines 810 are activated and thus one layer of a structure 812 isexpanded into the flow cell volume 806 between a substrate 804 and afluidic structure 802 sequentially, forcing beads 808 out from a levelexceeding a one to one correspondence with sensors, by displacing excessbeads 808 as the control lines 810 are activated. The beads 808 may thenbe held in place during sequencing cycles. As can be seen in FIG. 8,there is sufficient room for liquid flow below the control lines, butinsufficient room for bead movement. When a set of sequencing cycles hasbeen completed, the control lines may be deactivated, and the beads 808may then be removed by fluid flow through the sensor array region.

In an alternative embodiment, the number of beads may be lower than thenumber of sensors. The number of beads may be close to the number ofsensors, with control lines being activated such that beads are causedto be localized with sensors. Said localization may be assisted byalternating flow directions in the sensor array region, introducingvibrations or oscillations, or the like, such that the beads areundergoing frequent motion, until such time as motion is prevented bythe control lines being sufficiently activated that said control linesare too low to permit beads from being able to move from one sensorregion to another. Further movement of the beads combined with furtheractivation of the control lines will serve to more completely center thebeads over the sensors.

In a further alternative embodiment, a structure having a shape similarto that shown in FIG. 8 may be molded, machined, or otherwise formedsuch that the shape is similar to that which will occur wherein thecontrol lines are fully activated. Said structure may be slowly loweredover the bead covered sensor array.

In yet a further embodiment, in order to cause a higher percentage ofsensors to have associated beads, beads may be attached to sensors bybiotin streptavidin binding thiol binding or the like after a set ofbeads has been localized by one of the aforementioned structures.Additional beads may then be introduced into the sensor array region,and the process repeated. If binding agents are localized to areas abovesensors, as may be done as previously described, any excess beads whichare caught or pinched by the structure will not bind, and may be washedaway before beginning the sequencing cycles.

In yet a further embodiment, a significant excess of beads may beintroduced into the sensor array region. A single control line at anexit from the sensor array region may be activated to trap the set ofbeads. The aqueous conditions and/or temperature may be changed topermit binding of beads to the sensors. The excess of beads may then beremoved by releasing the control line allowing flow of the beads out ofthe sensor array region with an aqueous reagent.

Some embodiments combine pH sensing with electrochemistry detection as aresult of the incorporation of a reversibly reducible layer which may befabricated above the previous sensor design. Such sensors are availablefrom Senova Systems. During a sequencing cycle, a reducing reaction willoccur if a base has been incorporated in the bead associated with asensor. The level of reduction can be measured, and after the completionof the sequencing cycle, a voltage can be impressed on the sensor,causing an oxidation of the surface, returning it to its original state,whereupon it can be used for the next sequencing cycle.

In some embodiments, magnetic beads are used without a magnetic array.The magnetic beads self assemble into a monolayer with uniform spacing,the spacing of which may be influenced by the use of an external magnetto change the local field strength. Said magnetic beads may be caused tobe separated at a spacing which matches the spacing of a sensor array,and then may be caused to bind to the sensor array by changing aqueousconditions, temperature or the like as previously described. Slowtranslation or movement of the beads may be appropriate after binding inorder to enable alignment of the beads with the sensors. Suchtranslation or movement may need to occur in multiple dimensions, whichmay include x, y, theta, and spacing. The addition of depressionsassociated with the sensors in a one to one correspondence may result inbetter alignment between the beads and the sensors permitting betterdetection.

FIG. 11 illustrates various embodiments, where the magnetic,paramagnetic, non magnetic particles may be of shapes other thanspherical for use with either a sensor array with magnetic retention(1102), a sensor array with electrical confinement, or a sensor arraywith self assembled particles. The particles may be planar, round,rectangular (1104), star shaped, hexagonal (1106), or other shape. Inother embodiments, the particle may dendritic, enlarging the surfacearea of said particle. Said dentritic particle may be generallyspherical, planar, oval, or any other shape. In yet other embodiments,said particle may be porous; if said particle is porous, the pore sizemay be of sufficient size as to permit free movement of DNA, polymerase,dNTPs and other moieties necessary for primer extension sequencing orother applications as appropriate.

In another embodiment, not requiring a magnetic bar array, the beads maybe replaced with DNA balls, created by using rolling circleamplification. The DNA balls may then be digested by a DNA nuclease. Inanother embodiment, the balls may fabricated from a monomer or polymer,such as polystyrene, which may be dissolved subsequent to sequencing,using an organic solvent such as acetone, thus freeing the attached DNAby the same process, both of which may then be removed from the flowcell by flowing a reagent through said flow cell.

In yet another embodiment, the bead may be held in place by a bindingbetween moieties attached to the well and moieties attached to the bead.The well may be shorter than the radius of the bead, and may have ashape other than circular, so that reagents may flow around the bead,while the bead may be bound at several points at the entrance to saidwell. The binding may be a Streptavidin Biotin binding, a DNA DNAbinding, a DNA PNA binding, a PNA PNA binding, Thiol Au binding,photoactivated binding, covalent binding, or the like. Said binding maybe released by raising the temperature, or by introducing a reagentwhich reduces the affinity between the moiety on the well and the moietyon the bead, permitting the beads to be removed from the flow cell byflowing a reagent through said flow cell. The beads may be furtherinduced to move from the well in which said bead has been bound bysonicating the bead and well structure. Sonication may be done at thesame time that a reagent is flowed through the flow cell in order toremove said beads from said flow cell.

In the process of amplifying DNA in chamber free system as described inprovisional application 61/491,081, various factors may be potentiallysubject to optimization. Among these include the frequency, voltage andsize and shape of the confinement “cell” used to confine the polymerase,target DNA and generated amplicons. If confinement were the onlyconsideration, it would be possible to confine almost any size ofamplicon, without regard to the small size of said amplicon. However, inorder to be able to have a field strong enough to insure properconfinement, the field may prevent proper activity of the polymeraseincorporation of bases during the PCR or isothermal amplificationprocess, or may pull the polymerase and or extended primer from thecomplex of the target DNA extended primer and polymerase. In oneembodiment, it is desirable to optimize a combination of frequency,voltage and size of the confinement cell, depending on the size of theamplicon.

In some embodiments, the amplification reaction is inverse PCRamplification, hot start PCR amplification, methylation specific PCRamplification (MSP), nested PCR amplification, reverse transcriptionreaction, reverse transcription PCR amplification (RT-PCR), TouchdownPCR amplification, intersequence specific PCR amplification (ISSR-PCR),co-amplification at lower denaturation temperatures (COLD-PCR), solidphase amplification, bridge PCR amplification, or single primer bridgeamplification.

In some embodiments, the amplification reaction is helicase dependentamplification (HDA), a nicking enzyme amplification (NEAR), recombinasepolymerase reaction (RPA), transcription mediated amplification (TMA),self-sustained sequence replication (3SR), nucleic acid basedamplification (NASBA), signal mediated amplification of RNA technology(SMART), loop mediated isothermal amplification of DNA (LAMP),isothermal multiple displacement amplification (IMDA), solid phaseisothermal, bridge isothermal, single primer isothermal amplification(SPIA), circular helicase dependent amplification (cHDA), or rollingcircle amplification.

In generating a DC field for electrophoretic concentration orconfinement, electrolysis products can build up. These include hydroniumand hydroxide ions. To minimize effects from these ions the DC field canbe pulsed so the net DC is much lower. In some embodiments the pulseduty cycle can be reduced after the DNA has migrated closer to thecenter electrode. In other embodiments the process can use DC toconcentrate the DNA and then use AC to maintain concentration orconfinement of said electrolysis products. In other embodiments both DCand AC can be used for concentration and confinement.

In some embodiments, pulsed field gel electrophoresis is used. Forexample, a non-sinusoidal AC waveform may be used, where a higherpositive voltage may be balanced by a longer but shorter negativevoltage such that the average voltage is substantially zero. The higherpositive voltage may be used with a polymer concentration, such thatreptation of the DNA occurs in the polymer solution. The polymersolution may effectively cause a lower migration of DNA in the directionof the lower field compared to the migration in the higher strengthelectric field, which may thereby increase the mobility of the DNA. Inthis manner the DNA may migrate more in the desired direction whileother molecules such as Mg move freely back and forth due to thebalanced nature of the AC waveform. The migrational variance may also befrequency dependant, so that different sizes of DNA may be captured. Thepulsed field gel electrophoresis may be 1D or 2D, and may use contourclamped electric field, transverse alternating field electrophoresis, orrotating gel electrophoresis, any of which may be used with either agel, an entangled polymer, or another sieving matrix.

In generating a dielectrophoresis field, typically a sinusoidal waveformis used. While this may be ideal for an application intended strictlyfor confinement or separation of different species, it may cause issuesfor a system where a biochemistry reaction may be performed within theconfinement volume. For example, high fields are likely to result inlocalized heating. In one embodiment, a modified sinusoidal waveform mayinstead be used. For example, the modified sinusoidal waveform may havethe voltage removed at the top of the sinusoid, or at any other point inthe sinusoidal waveform, allowing localized diffusion, permittinghybridization of the amplicons to primers, binding of polymerase to theduplex DNA, and binding and incorporation of nucleotides or nucleotideanalogs. The field may then be reinstated after an appropriate period oftime. The same process may occur at the peak with the opposite sign inthe modified sinusoidal waveform. In other embodiments, any otheralternating current waveshape can be used for concentration orconfinement. Alternatively, the interruption in the sinusoidal waveformmay occur only once per cycle, or may occur once in every severalcycles, or once in many cycles, so that any “stray” amplicons may becaptured in the regions with lowest field strength and returned to themain volume of the confinement volume. Alternatively other wave formssuch as square, trapezoidal, non symmetrical wave forms, etc may beused.

In some embodiments monoclonal beads may be generated in a smallmicrofluidics device. In one embodiment the electrodes and magnets maybe fabricated on a thin sheet wherein a top surface may be bonded orfused in place creating an integrated microfluidics device. Themicrofluidics device may have electrical and fluidic connections made toit. The microfluidics device may then be placed in good thermal contactagainst a first heated plate, for example by vacuum or air pressure. Asecond plate may be situated above at a different temperature. The twotemperatures may be chosen to facilitate PCR amplification. After onetemperature point is complete the card may be transferred or caused tocome into contact with the upper heated plate, for example by vacuum orair pressure. Because only the thin card and the reagents in the cardneed to change temperatures the system can have fast temperaturetransitions, and consume minimal power.

In other embodiments that provide a minimal thermal mass, the electrodesbuilt into the amplification microfluidics device may be used asresistive heaters to locally heat the liquid. In some embodiments thechange in resistance of the electrodes are used to measure thetemperature for better thermal control. In other embodiments thesensors, such as the NanoBridge or NanoNeedle (described herein) areused as a temperature sensor for better control of the area of interest.

In some embodiments, it may be desirable to perform DNA amplificationfrom a single copy of DNA. If a polymerase error is made inamplification at an early point in the amplification process, such as inthe first cycle in a PCR amplification, the error will proliferate suchthat it may not be possible to differentiate between the correctsequence and the error sequence. Thermostable polymerases typically havemuch higher error rates than mesophilic polymerases or thermophilicpolymerases which may be not suitable for PCR as they inactivate duringthe denaturation step of PCR. Thus in some embodiments, it is desirableto use a highly accurate polymerase for the initial portions of a PCRreaction, where the highly accurate polymerase is not sufficientlythermostable to prevent inactivation during PCR, but may provide betteraccuracy than a more thermostable polymerase. The highly accuratepolymerase may have a low K_(off), such that the highly accuratepolymerase is substantially bound to the active extension site, in thepresence of other polymerases which may be sufficiently stable toprevent significant inactivation during the denaturation steps of a PCRamplification.

In some embodiments, the highly accurate polymerase is introduced to avolume with primers and template prior to the introduction of the morethermostable polymerase. In other embodiments, the thermostablepolymerase is heat activated, such that any heat activated polymerasewill be inactive for the first cycle(s) of the PCR.

In other embodiments a combination of isothermal and PCR amplificationreactions is used. Initial amplification may be performed by a highlyaccurate non thermal stable polymerase, and subsequent amplification maybe performed by a less accurate thermal stable polymerase that is notsubstantially inactivated by the denaturation step of PCR.

In an alternative embodiment, a clonal population is generated in thearea of individual sensors in a sensor array. The sensors may beNanoNeedles or Nanobridges or other sensors to detect the event ofpolymerization. In one embodiment, primers are preferentially attachedto the surface of the sensors. The primers may be preferentiallyattached as a result of a difference in materials, where the material ofthe sensor is more advantageous for attachment then the areas betweenthe sensors of the sensor array. In an alternative embodiment, a maskmay be applied to areas between the sensors of the sensor array, and asurface modification may then be performed. Subsequently, the mask maybe removed; leaving an area between the sensors of the sensor arraywherein the surface modification has not been performed. The surfacemodification may include attachment of biotin, applying a layer of goldand various other methods as are known in the art.

Primers may then be preferentially applied to the areas on the surfacesof the sensors in the sensor array. In one embodiment, the primers areattached as a result of a biotin streptavidin binding, where thestreptavidin is attached to the 5′ end of the primers. In anotherembodiment, a thiol group may be attached to the 5′ end of the primers,which can then bind to the gold layer previously applied above thesensor, forming an Au—S bond. If a PCR reaction is desired, the primersmay be modified with DTPA such that two thiol gold bonds are formed,preventing the dissolution which may otherwise occur from the 60 to 95Ctemperatures routinely used in PCR.

After a set of sequencing cycles is completed, the primers are removedand replaced. Buffer conditions can be changed to weaken the biotinstreptavidin bond, such as high concentrations of GuHCl at low pH;alternatively the temperature can be raised to over 70 C to break thebiotin streptavidin bond. Thiol bonds can likewise be broken at elevatedtemperatures. Aggressive means may be used, as damage to the polymeraseand DNA is no longer consequential. In one embodiment, organic reagentsare used to break the binding between the extended primer and thesurface, such as a covalent binding. After the extended primers areremoved, new primers may be flowed into the volume above the sensors,enabling the device to be used again for another set of sequencingcycles on a another set of DNA samples.

In some embodiments as schematically depicted in FIG. 12, the sensorarray may have elements 1200 that are provided with an additional arrayof electrodes 1206, which may be used to perform dielectrophoreticconcentration. Dielectrophoretic concentration may be initiallyperformed to attract sample DNA dNTPs, and primers to each sensorregion. Amplification can then commence in the region of each sensorwhere a sample DNA molecule is located. During the amplificationprocess, the dielectrophoresis forces may also aid in preventing crosscontamination between different sensor regions undergoing amplificationby retaining amplicons. In order to insure that polyclonal regions arenot generated, the concentration of input DNA needs to be low enoughthat most sensor regions have one or zero sample DNA molecules. DNAsamples could be single stranded or double stranded depending on theamplification methodology. The amplification reaction can be either aPCR reaction, or an isothermal reaction. In some embodiments theadditional electrodes 1206 are shown as having the same voltage relativeto voltage level of the sensors. In an alternative embodiment electrodeson either side of a sensor may have voltages of opposite sign relativeto each other.

Said sensor array elements 1200 may be fabricated on a substrate 1212,and may have magnets 1216 utilized for retention of magnetic orparamagnetic particles or beads 1202, wherein said magnetic orparamagnetic particles or beads may be held down against an electrode1204, an against a dielectric 1210 and or upper electrode 1208.Detection may utilize said electrode 1204 and said upper electrode 1208,while dielectrophoretic concentration/confinement may utilize electrode1204 and outer electrode 1206, wherein said outer electrode may comprisea single electrode or may comprise multiple electrodes.

Amplification may be a solid phase amplification, where one primer is onthe surface of the bead, and a second primer is in solution, or theamplification may be solid phase where all primers are on the bead.Alternatively, the amplification may be performed where both primers arepresent in solution, and one primer, or both primers are also present onthe bead.

The electrode configuration may take various different forms, includinga planar electrode on both major planes of the flow cell, or there maybe one electrode on the surface opposite the beads, and a set of smallerelectrodes associated with each detector location.

FIG. 12 illustrates the use of the amplified regions above the sensorsin the array of sensors in a sequencing reaction. After theamplification reaction has been completed, the volume above the sensorarray may be washed, removing amplicons, polymerases, and dNTPs.Polymerase and individual dNTPs may then be flowed into the volume abovethe sensor array, permitting binding, incorporation, and detection ofthe incorporation events, resulting in the determination of the sequenceof the different amplified sample DNA molecules.

In some embodiments, the sensor is used for several purposes, such as,for example, detecting the presence of beads when introducing beads,detecting amplification associated with the bead (e.g., real time PCRamplification or end point PCR amplification), and detecting sequencingreactions.

When generating clonal beads a large percentage of the beads will haveno DNA template. In addition others may have poor amplification. Thesebeads do not provide useful sequencing so it is desirable to removethese beads to improve instrument throughput and reagent utilization. Insome embodiments, beads with no or minimal amounts of template areseparated using an electric field. The beads on which amplification hasoccurred have more fixed negative charge from the amplified DNA and canbe separated from those, on which amplification has not occurred, by theuse of an electrophoretic separation. This permits the situation asshown in FIG. 3 where most positions in the magnetic array are depictedas being occupied by beads which have had an amplification reaction, andare therefore suitable for use in a sequencing reaction.

Beads fully loaded with templates have a higher charge so will movefarther in an electric field than beads with only primers or fewtemplates. In one embodiment as shown in FIG. 13A, FIG. 13B and FIG.13C, this separation is done in a flow through module. A first fluidicinput 1311A allows the injection of mixed beads. A second inlet 1312Aallows the injection of a buffer solution without beads. A first outlet1311B is downstream from the first inlet 1311A. A second outlet 1312B isdownstream from the second inlet 1312A.

The fluidic flow rates can be set by fluidic resistance or pumping speedsuch that more liquid flows in the second inlet. In the embodiment shownin FIG. 13B, the inlet and outlet widths may be varied to createdifferent fluidic resistances, but other methods of modifying thefluidic resistance such as different length or height may beanticipated, or the use of flow restrictors in parts of the systemexternal to the enrichment module 300. Similarly the fluidic resistanceof the first outlet 1311B and second outlet can be modified so moreliquid flows out the first outlet 1311B. In such a setup beads without asmall velocity perpendicular to the flow will exit the first outlet port1311B. Additional output channels can be added to facilitate separationof beads with medium levels of template. In some embodiments, the flowrate in each output channel may be controlled directly by providingindividual pumps for each outlet channel.

A pair of electrodes 1313, may be provided which enable generation of anelectric field perpendicular to the fluid flow in the separation section1310 such that the template loaded beads, which may be brought into theenrichment module 1300 through inlet 1311A while an additional reagentmay be introduced to the module through a second input 1312A, maymigrate out of the flow path towards second outlet 1312B. Fluidic ports1309 allow connection to the system plumbing.

The electrodes 1313 could be made of any electrode material compatiblewith electrophoresis. In some embodiments discrete metal wires may beused but metal traces may be also anticipated. Metals such as platinum,platinum/iridium, gold and other noble metals or alloys may beanticipated as well as corrosion resistant materials such as stainlesssteel. Non metal electrodes may be also anticipated.

In another embodiment the fluidic flow rates can be set by fluidicresistance or pumping speed such more or less fluid flows out the firstoutlet than the second outlet allowing steering of the bead flow stream.In yet another embodiment the bead stream could be adjusted by both theinlet and outlet fluid flows.

The flow through enrichment module 1300 can be constructed of nonconducting materials such as molded plastic, glass, ceramic or moldablepolymers such as PDMS, or of conductive materials which may be coatedwith a nonconductive coating, or combinations or these materials, or incombinations with other materials. Fluidic components can be fused,bonded, or held together with a clamp mechanism to create an enrichmentmodule including a separation section 1308. The enrichment module may inone embodiment include a molded upper piece 1308, and a flat substrate1302. In other embodiments, the enrichment module may be made of morethan two pieces, such as for example, three, four, five or morecomponents. If two components may be used, both sides may havenon-planar surfaces, such that fluidic or control channels may be formedin either component. If more components are used, any one of them can beplanar or shaped such that they include channels, depressions orprotrusions, or may be a combination of planar and shaped such that theyinclude channels, depressions or protrusions.

In some embodiments, the surface or a portion of the surface of one ormore components of the enrichment module has a zeta potential sufficientto cause significant electroosmotic flow. It may be desirable tominimize any mixing or turbulence which might otherwise result from saidelectroosmotic flow. In some embodiments, materials such as TiO², ZrO²,or BaTiO³ are selected such that the zeta potential and the resultingelectroosmotic flow are significantly reduced. In some embodiments, thezeta potential and the relationship between the zeta potential and achange in pH may change depending on a surface coating. In someembodiments the zeta potential may change significantly with changes ofpH, as is the case with the pH dependency of Silica. In other cases, thezeta potential changes very little with respect to pH, particularly inthe pH range from pH 7.5 to pH 9, as is the case with the pH dependencyof BaTiO³.

In other embodiments, surface coating(s) such as PEG (Poly EthyleneGlycol), methyl cellulose, n-dodecyl-B-D-maltoside, acrylamide,fluorinated alkane chains, PTFE, acrylate, or other cross-linked orpartially cross-linked polymers are used to modify the zeta potential,or combinations of surface coatings are used to similarly minimize theelectroosmotic flow. In some embodiments, polymers are used to fill theaqueous volume of the electroosmotic flow restriction section.

In other embodiments, a physical structure as shown in FIG. 13C is usedto reduce or eliminate unwanted mixing and turbulence due toelectroendosmotic flow. Such a structure may have a flow constrainingsection 1320, and said flow constraining section(s) 1320 may be used onboth sides of separation section 1308. Electric field may be distributedfrom electrodes (not shown) through buffer reservoir section(s) 1322.Said electrodes may be positioned in auxiliary input port(s) 1324, whichmay be used to bring buffer to and through the buffer reservoirsection(s) 1322 and flow constraining section(s) 1320. In an alternativeembodiment the electrodes are positioned in the buffer reservoirsection(s) 1322, with electrical connection to a voltage source whichmay be external to enrichment module 1300. Input beads and reagents maybe brought into said separation section 1308 through input ports 1311Aand 1312A, or reagents may be brought in through input ports 1311A and1312A, while beads are brought in through center input port 1326. Beadsmay be separated between output ports 1311B and 1312B.

In an alternate embodiment the electrodes are positioned in a bufferreservoir(s) separate from the structure shown in FIG. 13C with afluidic connection to allow current flow into the enrichment module. Insome embodiments, the buffer reservoir(s) allow electroendosmotic flowto more in one direction across the enrichment flow cell, rather thanhaving a circular flow with concomitant turbulence that would occur witha sealed flow cell, where any flow in one direction must be matched byflow in the opposite direction. In some embodiments the voltage can bestopped periodically to allow the fluid reservoirs to return to theirequilibrium state, where the liquid level in each reservoir is at thesame level, after electroosmotic pumping. In other embodiments, thevolume or cross section of said reservoirs is significant relative tosaid electroosmotic pumping, such that bead sets or multiple bead setsmay be separated or enriched prior to allowing said fluid reservoirs toreturn to their equilibrium state.

In other embodiments the zeta potential magnitude is reduced byprotecting the silanol groups with a compound such astrimethylchlorosilane which decreases the number of ionizable silanolgroups. FIGS. 13D and 13E microphotographically illustrate the use of astructure as shown in FIG. 13C, where a stream of input beads 1314 isillustrated without a field applied in FIG. 13D, and thus all inputbeads 1314 are carried to output 1311B as if they were lower chargedbeads 1314A, and no beads appear to be higher charged beads 1314B pulledto outlet port 1312B. The flow constraining section 1320 shown in FIG.13C is shown in more detail, showing the fluidic passages 1316, andsupport columns 1318. In FIG. 13E, where a field is applied to saidseparation section 1308, and said stream of input beads 1314 isseparated into low charged beads 1314A which is carried to output port1311A, and higher charged beads 1314B are pulled and carried to outputport 1312B.

In some embodiments, the thickness or depth of the separation section1308, fluidic inputs 1311A, 1312A, fluidic outputs 1311B, 1312B,separation section 1308, electroendosmotic flow restriction section(s)1320, and buffer reservoir(s) 1322 may be the same. In otherembodiments, the thickness or depth may be different for differentsections, for example, the thickness of the electroendosmotic flowrestriction section(s) 1320, may be less than that of the separationsection 1308 or the buffer reservoir(s) 1322.

In some embodiments, the thickness or depth of the separation section308, and other fluidic sections of the enrichment module is from 10 to1000 μm; in other embodiments the thickness or depth of the enrichmentmodule is from 20 to 200 μm, 50 to 150 μm, 200 to 500 μm, or from 70 to130 μm.

In some embodiments the width of the flow restrictors in theelectroendosmotic flow restriction section(s) 313 is from 10 to 1000 μm,in other embodiments, the width of the flow restriction is from to 20 to200 μm, 50 to 150 μm, 200 to 500 μm, or from 70 to 130 μm.

In some embodiments the length of the enrichment zone can be up to 2 mm,2 mm to 10 mm, 10 mm to 100 mm. In some embodiments the width of theenrichment zone can be up to 1 mm, 1 mm to 4 mm, 4 mm to 10 mm and 10 mmto 100 mm.

In some embodiments, the enrichment module 1300 has a feedback system(not shown) to compensate for varying charge levels that may existbetween different batches of beads. Such a feedback system may thenpermit the electrophoretic voltage to be automatically tuned for aparticular batch of beads, and to be automatically retuned for asubsequent batch of beads. Said feedback system may use reflected light,absorbed light, deflected light, fluorescent light, capacitive couplingto the beads, direct conductivity detection of the Debye layerassociated with the beads, ISFET/ChemFET detection of the beads, or mayuse any other appropriate detection means. Said detection means may beconfigured such that detection of beads is effectuated in or associatedwith one or more fluidic outputs 1311B and 1312B, or may be configuredsuch that detection of beads is effected in or associated with theseparation section 1308.

In some embodiments the feedback system is used during a separation of abatch of beads, tuning the flow rate and electoosmotic voltage so thatthe beads are optimally separated and flow into the nominal desiredposition in each output 1311B and 1312B. Said flow rate control may beeffectuated by the use of a variable pressure applied to said flow, avariable vacuum applied to said flow, a variable restriction in the saidflow, or any combination thereof.

In some embodiments, the bead slurry may need to be concentrated. In oneembodiment the bead solution is passed by a magnet to hold the beads. Byremoving the magnetic field or using a higher flow rate the beads can bereleased in a more concentrated form.

In some embodiments the enrichment module 300 is used to separatenegatively charged DNA from proteins, including cellular membranefragments which may be comingled with said DNA after lysing of thecell(s) from which said DNA and said proteins and said cellular membranefragments may have originated. In some embodiments it may be desirableto separate the DNA from proteins, most of which may be positivelycharged, and to also separate the DNA from proteins which may benegatively charged at neutral pHs, such as human serum apotransferrin,thyroglobulin, or BSA. Such proteins which may be negatively charged atneutral pHs typically have pKa values above 4.0, whereas the pKa for DNAis 1.0. The electrophoretic mobility of proteins is typically much lowerthan that of the highly negatively charged DNA, permitting easyseparation of DNA in an enrichment module 300. Such separation may beperformed at a low pH such as a pH below 7, a pH between 6 and 7, a pHbetween 5 and 6, a pH between 4 and 5, or a pH between 3 and 4,permitting the enrichment module to run below the pKa of said proteins,and above the pKa of the DNA.

In some embodiments, the DNA may be dielectrophoretically captured afterbeing substantially separated from any proteins and cellular membranes.Said dielectrophoretic capture may be effected in a fluidic outlet 311B,312B, or may be effected in a separate module. After saiddielectrophoretic capture, the buffer may be changed, for example from alow pH as previously described to effect a separation from proteins orcellular membranes, to for example, a buffer suitable for PCR,isothermal amplification, or sequencing, where the pH is approximatelyoptimal or otherwise suitable, for example, for polymerase activity.

The voltage applied to the electrodes 1313 can be reduced or evenreversed periodically if necessary should beads stick to the electrodes.The voltages used may be greater than that required for electrolysis(1.23V at 25C at pH 7), or may be less than the voltage needed forelectrolysis. Higher voltages and narrower gaps provide a higher fieldstrength and more force on the beads. The voltage on the system can becalibrated by flowing beads without or with limited template and settingthe voltage and or flow rate such that these beads may be not moved farenough to enter the second outlet while beads with template may bedirected into the second outlet.

Non-flow-through enrichment modules may be also anticipated. In oneembodiment beads are introduced to a chamber and a magnetic field orgravity pulls the beads down. An electric field is established pullingthe beads with template up. In some embodiments a capture membrane orfilter can be added in front of the positive electrode to facilitateconcentration of the beads.

In one embodiment, beads are removed from the flow cell as a result ofactions and methods which are performed within the same instrument wherethe flow cell is used to sense a reaction, such as a sequencingreaction.

In another embodiment, the flow cell assembly is removed from theinstrument where the flow cell is used to sense a reaction, such as asequencing reaction, and moved to another instrument or device, wherethe beads are removed from the flow cell.

In yet another embodiment, the flow cell assembly is removed from theinstrument where the flow cell is used to sense a reaction, such as asequencing reaction, and shipped to a central refurbishment site, wherethe beads are removed from the flow cell.

In another embodiment, the flow cell assembly is removed from theinstrument where the flow cell is used to sense a reaction, such as asequencing reaction, and moved to another instrument or device, wherecoatings may be applied, removed and/or reapplied to the flow celland/or fluidics manifold.

In yet another embodiment, the flow cell assembly is removed from theinstrument where the flow cell is used to sense a reaction, such as asequencing reaction, and shipped to a central refurbishment site, wherecoatings may be applied, removed and/or reapplied to the flow cell andor fluidics manifold.

In some embodiments, the flow cell and/or the fluidics manifold havevarious surface coatings. Such surface coatings are used to reducenonspecific binding of moieties in the various reagents, to the surfacesin said flow cell or fluidics manifold. In some embodiments, thecoatings intended to reduce nonspecific binding may include PEG(Polyethylene Glycol), BSA (Bovine Serum Albumin), PEI(Polyethylenimine), PSI (Polysuccinimide), DDM(n-dodecyl-b-D-maltocide), fluorinated coatings, Teflon coatings,silanization coatings, or other appropriate coatings.

In one embodiment, the coatings are applied, removed and/or reapplied tothe flow cell and or fluidics manifold as a result of actions andmethods that are performed within the same instrument where the flowcell is used to sense a reaction, such as a sequencing reaction.

In some embodiments the sensor combines pH sensing with electrochemistrydetection as a result of the incorporation of a reversibly reduciblelayer which may be fabricated above the previous sensor design. Suchsensors may be available from Senova Systems. During a sequencing cycle,a reducing reaction will occur if a base has been incorporated in thebead associated with a sensor. The level of reduction can be measured,and after the completion of the sequencing cycle, a voltage can beimpressed on the sensor, causing an oxidation of the surface, returningit to its original state, whereupon it can be used for the nextsequencing cycle.

As shown in FIG. 9, in some embodiments, a redox reaction may beperformed where the redox potential may comprise a combination of an ACpotential 904 combined with a nominal DC potential 902, where thenominal DC potential 902 is one half of a sine wave, starting at zerovolts, rising to a maximum, and returning to zero volts, where said ACpotential 904 may be superimposed on said nominal DC. The AC potential904 waveform may be of a frequency which is 10 times as high as thenominal DC potential 902, or AC potential 904 waveform may be of afrequency which is 10 to 100 times as high as the nominal DC potential902, or AC potential 904 waveform may be of a frequency which is 100 to1000, 1000 to 10,000, 10,000 to 100,000, 100,000 to 1,000,000, 1,000,000to 10,000,000 times as high as the nominal DC potential 902. The ACpotential 904 waveform may be a sinusoidal waveform, a triangularwaveform, a square waveform, or any other sort of symmetrical orasymmetrical waveform. The DC potential waveform may be half of a sinewave, an isosceles triangular waveform, a saw tooth waveform, or anyother waveform starting at zero volts and increasing to maximum andthence returning to zero volts. The magnitude of the superimposed ACpotential 904 waveform may be constant, or may change during the DCpotential 902 waveform, for instance, the AC waveform may be smallerwhen the DC waveform is close to zero, and the AC waveform may increaseas the DC waveform reaches its maximum potential. The current 906resulting from the combination of the AC potential 904 and nominal DCpotential 902 may be a non linear function of applied potential.

In one embodiment where the quantity of data which is collected isminimized, it may be desirable, in addition to increasing the speed ofthe reaction, to align the active regions with the normally occurringrectilinear nature of semiconductor electronics. In previous systems,the locations of the active reactions may not align well with an arrayof detectors. It is preferable to arrange the detector electronics in astrict rectilinear fashion, as opposed to convention which bringsreagents in and out from the corners 1404A and 1404B of a chip 1400 asshown in FIG. 14. This approach both prevents alignment with the reagentslug, and wastes a significant area of the chip, as the reagent slugcannot flow well (or at all) to the other corners of the chip 1402, aswell as causing nonuniformity of flow (and loading efficiency 1406) dueto the large difference in cross sectional area from the center of thechip to the inlet and outlet ports in the corners of the chip 1404A and1404B.

In one embodiment, multiple fluidic inlets 1512A as shown in FIG. 15 maybe used in a multiple flow cell sensor device 1500, permitting greaterutilization of the chip area, and more uniform and aligned reagent flowrelative to the chip and said chips readout structure. In someembodiments, samples may be introduced at different times to thedifferent channels of the flow cell, permitting different samples to beused without the need for bar coding or other means for sampleidentification. The multi flow cell sensor device may have multiple flowcells 1510, with an input port 1512A an output port 1512B, and a line towaste 1516, for each flow cell, and wherein each input and output portmay be configured so as to optimize uniformity of flow across the sensorarray within said flow cell 1510, potentially having angled side wallsand or surface roughness so as to optimize said flow uniformity. Saidflow cells may further be configured with valves 1504 and controls forsaid valves 1506 for controlling the flow of samples and or otherreagents in immediate proximity to the flow cells 1510. The samples andother reagents may be brought in using input ports 1502.

In some embodiments, the samples are introduced different times in aprocess or set of processes. For example, a second set of beads thathave undergone an amplification reaction and consequently have extendedprimers are introduced to a channel of a flow cell associated with achip, where another channel of the chip may already have a first set ofbeads. The first set of beads may have already undergone anamplification reaction and consequently have extended primers containedtherein, and the channel with the first set of beads may have beenexposed to a sequencing cycle, or may have been exposed to a number ofsequencing cycles. In another embodiment, the second set of beads isintroduced to the same channel where the first set of beads iscontained. In another embodiment, wherein amplification and sequencingmay be performed in a single area of an array as described elsewhereherein, a set of beads in one channel may be undergoing an isothermalamplification, while a second set of beads in another channel may beundergoing a sequencing reaction.

In some embodiments, wherein a temperature change may be needed for afirst process but not for a second process, the second process may betemporarily halted or paused while the temperature change occurs, andmay then commence after the temperature is returned to the previoustemperature. For example, an amplification reaction process may need tomelt off and remove a second strand after primers associated with a beadhave been extended, so that complementary unextended primers can behybridized to the primers associated with the bead, so that a sequencingby synthesis reaction may commence. Prior to raising the temperature ofthe chip with multiple channels, the channel having a set of beadsundergoing a sequencing reaction may pause the sequencing reaction, andmay actuate the co-localized virtual nanoreaction well electrodes. Inthis manner, hybridized partially extended primers are kept localizedwith each virtual nanoreaction well, while the extended primers notassociated with beads in the channel, may be dissociated from theprimers associated with the beads, and then removed from the channel.

The system could divide the samples over multiple fluidic channels orchips if they are too large, or combine the samples if they arecombinable (for example barcoded samples). In some embodiments samplesprovided to the instrument would be ready for sequencing. In otherembodiments samples could be processed by the instrument to generatesequencing ready samples.

In another embodiment, multiple input ports to a single flow cell orelectro-wetting are used to introduce samples to portions of a flowcell, permitting more samples to be used at a time, without risk ofcross contamination.

In another embodiment, an electro-wetting system may be provided to movereagents within a part of the flow cell without generating a reagentinterface, thus completely preventing any mixing of reagents prior tothe reagent reaching a desired portion of the flow cell, and may thusprovide an extremely sharp transition in reagents.

In some embodiments, it is desirable to have a sharper transitionbetween one reagent and another, for example, when flowing a dNTPcontaining reagent through the flow cell where the sensors arepositioned. This may help to provide a quicker transition between aconcentration where essentially zero dNTPs are present, to a reagent aconcentration at a desired dNTP for incorporation by polymerase toextend, or further extend the primer. This may enable a shorter timebetween a time of initiation of base incorporation and a time where asignificant percentage of the primers or extended primers have had thenewly introduced nucleotides incorporated into appropriate locations indifferent colonies in the flow cell. The shorter time may allow agreater change in the signal level of a sensor or sensors per unit time,which may improve the signal to noise. Electronic sensors are typicallysubject to having noise, which may include thermal noise and flickernoise, both of which can be minimized by reducing the time intervalduring which an integrated signal is sensed by a sensor.

This quick transition is drawn in contrast with a system where a slowertransition occurs between a concentration where essentially zero dNTPsare present, to a reagent with a concentration at a desired dNTP forincorporation by polymerase to extend, or further extend the primer.This slower transition may occur as a result of diffusion of dNTPs froma reagent solution containing dNTPs into a reagent solution whichinitially has essentially zero dNTPs. This may occur while transitioningthrough a long channel through which reagents are flowed prior tointroduction into a flow cell where sensors are positioned for thedetection of a sequencing reaction or another reaction. Further mixingmay result from changes in channel width, corners through which thereagents are flowed, irregularities in the surface of a channel throughwhich reagents are flowed, slow flow through channels through whichreagents are brought to a flow cell, or slow flow through a flow cell.

In one embodiment, the channel length may be substantially reducedbetween a point in the fluidics system where a transition between aconcentration where essentially zero dNTPs are present, to a reagentwith a concentration at a desired dNTP for incorporation by polymeraseto extend the primer is generated, and the point in a fluidics systemwhere the reagents are introduced into a flow cell. Said channel lengthbetween the generation of a transition between said reagents and saidflow cell may be a micron or less, one to five microns, five to 20microns, 20 to 100 microns, 100 microns to 300 microns, 300 microns toone millimeter, one millimeter to three millimeters, three millimetersto ten millimeters, or ten millimeters to 30 millimeters. In general theshorter the distance between a position in the fluidics system wherein atransition between said reagents is generated and a position whereinsaid reagents are introduced into said flow cell, and the fewer thenumber of transitions in the size of flow cross section of the fluidicssystem, or corners in said distance, the lower the diffusion of dNTPs orother moieties will be, and thus the shorter the time frame will bebetween a reagent with a concentration of essentially zero dNTPs and areagent with a reagent with a desired concentration of dNTPs at eachsensor in a flow cell.

Minimizing the time for the polymerization reaction may also improve thesignal to noise associated with the detection of the polymerizationreaction. The noise associated with the detector may be fairlyconsistent with time, and the total integrated amount of signalgenerated may be the same regardless of the period of time over which itis generated. Thus reducing the time over which the data may be taken byspeeding the time associated with binding of the polymerase and/or byproviding a quick transition in the concentration of the dNTPs as theyare introduced in a reagent slug can minimize the amount of noise whichmust be dealt with in analysis. As a result, the noise bandwidth whichmust be dealt with by the analysis software may be reduced.

In some cases, where the sensor is near an exit port, the number ofdNTPs needed for incorporation may be sufficiently large that depletionof dNTPs may result in an increased time to generate the desired dNTPconcentration for incorporation by polymerase to extend the primer.Lower dNTP concentrations, longer distances with more colonies in a flowcell, larger colonies with more primers for extension may all result inan increased time to generate sufficient dNTP concentrations. In someembodiments, multiple input ports are used to provide inputs to a singleflow cell, to ensure the availability of dNTPs at each sensor and/orcolony. In some embodiments, reagent channels are provided above theflow cell, in additional layers of a PDMS liquid manifold. Duplicatesets of control lines, similar to those shown in FIG. 42 may beprovided, which may control duplicate sets of valves, similar to thoseshown in FIG. 42 and flow through duplicate sets of manifolds, similarto those shown in FIG. 42, which may then provide an alternative inputto the flow cell with a short distance between the point in the fluidswhere an interface between two reagents is created, and second orsubsequent input port to the flow cell.

A significant issue associated with next generation sequencing is theenormous quantity of data generated. Some systems can generate anaverage of 3000 or more data points for each useful base of sequencingdata. Storing and analyzing data adds significantly to the overall costof next generation sequencing. In some embodiments, data reduction isperformed in the simplest way, by acquiring less data. Polymeraseactivity can be significantly more rapid than the time needed to bringreagents with dNTPs completely through a flow cell; thus DNA coloniesclose to the inlet of a flow cell may have completely finished the nextsynthesis before the dNTPs have even reached the colonies near theoutlet of said flow cell. If data is acquired for the entire flow cellduring the time needed to detect reactions occurring anywhere in theflow cell, much of the data will be from regions of the flow cell whereno reaction is occurring. Depending on the time needed for the dNTPreagent slug to traverse the flow cell, and the speed of polymerization,most of the colonies in the flow cell will be either waiting for dNTPs,or will have completed their synthesis reaction, rather thanincorporating dNTPs and thus producing useful data.

In some embodiments, the readout of the detector electronics issynchronized with the movement of the reagent slug through the flowcell. A reagent slug containing dNTPs may initially enter the flow cell,but not yet move far enough into the flow cell for the dNTPs to bind andincorporate with any of the colonies. At this point in time it may bepossible to not take data at all. At a point slightly later in time thereagent slug will have entered the flow cell sufficiently to interactwith the set of colonies in the first region. At this point in time datamay be taken from the detectors associated with the colonies in a firstregion, but may be not taken for other regions of the flow cell. At asecond point later in time the reagent slug may have entered the flowcell sufficiently to begin to interact with the set of colonies in asecond region. At this point in time data may be taken from thedetectors associated with the colonies in the second region, and maylikely need to still be taken from the first region, depending on thespeed of the reagent slug and the speed of the polymerase, but may notneed to be taken for other regions of the flow cell. At a third pointlater in time the reagent slug has traversed through the flow cellsufficiently to begin to interact with the last set of colonies in theflow cell. At this point in time data may be taken from the detectorsassociated with the colonies in the last region. Some data may stillneed to be taken from previous regions, such as region immediatelypreceding said lat set of colonies depending on the speed of the reagentslug, the speed of the polymerase, the length of the flow cells, and thesize of the colonies, but may not need to be taken for other regions ofthe flow cell.

In other embodiments, time multiplexing with phase delay may beperformed to distinguish different samples from each other.

As the speed of valves used in a system may vary, and the size of tubes,channels and ports may vary from system to system and consumable toconsumable, the flow rate may also vary. In order to accommodate saidvariation, the first set of data taken from a system or consumable forwhich prior data may provide appropriate guidance as to the rate of flowin the system, more data may need to be taken, so that it may be assuredthat the data associated with a sequencing reaction is captured.

In one embodiment the timing of collection can be adaptively determinedfrom an earlier column, or from data from a previous cycle of the samecolumn. For example, if the typical detection event is occurring nearthe end of the collection time an additional delay may be added beforethe start of the next collection period of downstream samples. Similarlyif the typical detection event is occurring near the start of thecollection time the next collection period for downstream samples may bestarted earlier.

The size of the regions is shown in the illustrations as being onecolony wide, but the width of a region may be more than one colony wide.For example, if a flow cell is 1000 colonies wide from the inlet to theoutlet, a region may be a one colony wide, 10 colonies wide, 100colonies wide, 500 colonies wide, or any number in between. Thus theregion from which data is acquired at a time as the reagent slug movesthrough the flow cell may vary from one sensor, to tens of sensors, tohundreds of sensors wide, moving with the reagent slug as it traversesthe flow cell, and as the polymerization reaction completes.

The width of the region being read can be done at the time of readingthe chip, preventing the need to generate and discard data. This can bedone in a manner similar to that which may be used for reading a subareaof a CMOS image sensor, whereby a subset of the total rows or columnscan be read out at a time. Depending on the device structure, it may bepossible to select individual sensors, as it is possible to selectindividual pixels in some CMOS sensors. Alternatively, if the chipstructure is designed to read out a complete row at a time, usingseparate analog to digital converters for each column of the array ofsensors; the chip may be read out selecting which subset of rows aredesired. The subset of rows will change as the reagent slug progressesthrough the flow cell, and as areas of the flow cell complete thepolymerization reaction at the colonies in said area. In someembodiments, said separate analog to digital converters of comparatorsassociated with each column, wherein a counter may also be associatedwith each column, thereby allowing simultaneous conversion of the analogsignal into digital signals, while allowing more time, potentiallymultiple orders of magnitude more time for the analog to digitalconversion, with attendant improvements in signal to noise. In otherembodiments, the analog to digital converters may be successiveapproximation devices. In other embodiments, a pixel parallel readoutapproach may be utilized.

The width of the data region being collected needs to be large enough toaccount for a variety of factors to insure that all valid data is taken.These factors include variations in flow rate of the reagent slug, whichmay have slower flow near the edges of the flow cell due to interactionswith the surface. Other factors can include variations in thepolymerization speed due to concentration of the polymerase, variationsin concentration of the dNTPs, temperature variations, colony densityvariations, the number of repeats of the base being incorporated for acolony or colonies, amongst others. Any of these may require the widthof data which is being taken to be longer.

As described previously, dNTPs for extending the primer may be nativedNTPs, modified dNTPs which are incorporable by a native or modifiedpolymerase, or both native dNTPs and modified dNTPs. If a modified dNTPis used, the modification may act as a reversible terminator, a virtualterminator, or may change the charge of an incorporated nucleotide foreasier detection. Thus in some embodiments, the sequencing reactionincorporates all of the bases in a homopolymer run, or may incorporateone base at a time in a homopolymer run, reducing difficulties indetermining the number of bases in a homopolymer run when the number ofbases in a homopolymer run is large.

The kinetics associated with diffusion and binding of the polymerase tothe colony DNA may be noticeably longer than the kinetics associatedwith the diffusion, binding, and incorporation of the dNTPs. As aresult, the time period over which polymerization occurs may be longerif the polymerase is brought in the same reagent slug with the dNTPs, ascompared with bringing in a reagent slug with polymerase, followed bybringing in a reagent slug with dNTPs. Thus it may be advantageous tothe effort of minimizing the amount of data, to bring the polymeraseinto the flow cell, permitting the polymerase to bind to the colony DNA,prior to bringing in the dNTPs. If the polymerase is a processivepolymerase such that said polymerase is well retained between cycles,polymerase may be combined with the dNTPs to eliminate the need forseparate deliveries. In another embodiment, the delivery of a wash whichincludes phosphatase and polymerase may allow effective elimination ofresidual nucleotides and replenishment of the polymerase in a singlefluidic delivery. In other embodiments a reagent includes phosphatasewithout polymerase to effectively eliminate dNTPs by removal of thephosphate group via hydrolysis. The phosphatase may be shrimp alkalinephosphatase, calf intestinal phosphatase, or another phosphatase.

In general, for most clonal DNA sequencing systems, it is desirable tohave as much DNA as possible on a surface, in order to maximize theamount of data signal. However, as the DNA is randomly placed on thesurface, the spacing of the DNA may cause steric hindrance in asubsequent polymerization reaction.

In many different sequencing applications, target DNA or primers may bebound to the surface of the substrate. As a result of the attachmentmethods, the target DNA and primers may be randomly placed on thesurface, and may be in sufficiently close proximity that sterichindrance occurs during a polymerase extension. In some embodiments,primers are attached, bound, or associated with the substrate whilehybridized to DNA that overlaps the primer, which may provide a primingsite for a polymerase. Said primer and overlapping DNA may furthercomprise a polymerase, which may serve as a spacer to prevent binding ofsaid primers such that steric hindrance would occur, as for example whenthere is insufficient room for polymerase on each strand of DNA. Saidpolymerase and overlapping DNA may subsequently be removed so that atarget DNA may hybridize to said substrate attached, bound or associatedprimer for primer extension, which may be for amplification purposes orfor sequencing purposes. Alternately the primer could be in the form ofa hairpin with an extended end where the polymerase could bindeliminating the need for the longer DNA. Even if a Biotin Streptavidinbinding of the template is used, the size of the streptavidin (3 nm) maybe insufficient to properly space the DNA molecules such that there isroom for the polymerase (7 to 10 nm). In one embodiment, the target DNAmay be appropriately spaced such that steric hindrance cannot occur.This may be achieved by for example, using a complex with doublestranded DNA and a polymerase which is smaller, similar to or larger insize than the polymerase which will be used for the sequencing reaction.Alternatively, other proteins which bind to double stranded or singlestranded DNA and may be of appropriate size may be used, including apolymerase that is intended for a subsequent sequencing reaction. Thepolymerase may be processive, so that it remains bound during theattachment process, and may further have additional binding moietiesassociated therewith, to further enhance the ability of the polymeraseto remain in place during the DNA binding process. Alternatively,moieties other than proteins can used to space out the DNA and thenremoved resulting in DNA spaced to avoid steric hindrance. In someembodiments it may be desired that the surface of a substrate may not besaturated with DNA. A dsDNA strand is 20 Angstroms in diameter (2 nm),as opposed to a diameter of potentially greater than 100 Angstroms forthe polymerase. For example E. coli 22S RNA polymerase is 135 Angstromsin diameter (Kitano et al J. Biochemistry 1969 65 1-16); thus DNA spacedusing said polymerase may be spaced at a 2 percent (2̂2/13.5̂2*100)saturation level relative to dsDNA bound to said substrate in asaturated configuration. Polymerases do vary noticeably in size, forexample, B. strearothermophilus DNA polymerase I diameter was reportedto be 9 nm (Kiefer et al Nature Vol. 391 15 304) in contrast withpreviously cited 13.5 nm for E. coli 22S RNAP. In some embodiments, itmay be desirable to match the number of template copies to the size ofthe sensor; thus in some embodiments it may be desirable to use arelatively small number of template copies. In other embodiments, it isdesirable to use large numbers of template copies. Thus in someembodiments, it may be desirable to use 1,000 to 1,000,000 templatecopies, or 10,000 to 100,000 template copies, or 100,000 to 1,000,000template copies. In other embodiments, it may be desirable to use1,000,000 to 100,000,000 template copies, such as between 1,000,000 to5,000,000 template copies, 5,000,000 to 20,000,000 template copies, or20,000,000 to 100,000,000 template copies.

Steric hindrance may still occur when the spacing is equal to the sizeof the polymerase when the desired length of the DNA template is long,such that much if not most of said DNA is not perpendicular to thesubstrate. In some embodiments, the DNA template is sufficiently long to“ball up”, potentially taking up more space on the substrate than thepolymerase. In some cases a ssDNA and/or dsDNA binding moiety may beused to space DNA, where said DNA may be a DNA primer, and where saidssDNA and/or dsDNA binding moiety may be bound to other spacing moietieswhereby steric hindrance is minimized by the spacing and subsequentremoval of said ssDNA and/or dsDNA binding moiety and associatedadditional spacing moieties. Said spacing moieties may be otherproteins, or may be DNA of similar length to that desired for said DNAtemplate. In some embodiments, which may depend on the size of thedesired polymerase and the desired length of template, the level ofsaturation of attached DNA relative to saturated attached DNA may be0.001 to 40%, or may be 0.01 to 15%, or may be 0.1 to 5%, or may be 0.5to 2%.

In some embodiments, by using a polymerase or other spacer whileattaching, binding or associating DNA to a substrate, particularly apolymerase that is larger than the polymerase intended to extend saidsubstrate attached primer, steric hindrance will be significantlymitigated. In further embodiments, the polymerase may also be providedwith any other helper proteins or other moieties which may serve toincrease the effective size of the complex.

In some embodiments the Debye length of the read reagent solution issimilar to that of deionized water, which by definition has H⁺ and OH⁻concentrations of 10⁻⁷ molar, and a resultant Debye length of 680 nm. Inother embodiments, the ionic concentration of the read solution is aboutone micro molar, which results in a Debye length of about 300 nm. Thismay enable reading during a reaction. In other embodiments, the readsolution comprises ionic solvents which may be not wholly aqueoussolvents, permitting lower charge levels in solution, thus enabling alonger Debye length, and permitting the nanobridge (described herein) tosense more of a bead. For example, the read solution may permit sensingof a bead which has a diameter greater than one micron, such as two,three, five or more microns. A read solution which comprises nonaqueoussolvents may have a conductivity lower than that of distilled water,permitting a higher proportion of current to pass through the counterions associated with DNA as opposed to current which passes through thebulk solution. The solvents may be miscible with water, and may havesolubility of desired moieties; some representative solvents includeDMSO, alcohols, and ethers. Said miscible solvents may have lowerinherent ionic concentration, having a lower concentration of H⁺ and OH⁻than water. Said solvent may be used in conjunction with water, in partto provide a low concentration of hydrogen ions. In some embodiments,the ionic concentrations may be less than or equal to one micro molar,such as one micro molar to 0.5 micromolar, 0.1 micromolar to 0.5micromolar, 0.01 micromolar to 0.1 micromolar, 0.001 micromolar to 0.01micromolar, or less than 0.001 micromolar. In other embodiments, theionic concentration of the read reagents may be higher than one micromolar, such as for example, 1 milimolar, 2 milimolar, 5 milimolar, or ahigher concentration of ions. In some embodiments, the sensor may beable to detect changes in local charge, local conductivity, or localhydrogen ion concentration.

Many commercial buffers used for polymerization contain large amounts ofSodium or Potassium Chloride, which is not required for polymerization,and may further be heavily buffered. For example, the NEB IsothermalAmplification Buffer (1×), which is generally described as beingapplicable for Bst polymerase, contains 20 mM Tris-HCl, 10 mM (NH₄)₂SO₄,50 mM KCl, 2 mM MgSO₄ and 0.1% Tween-20; NEB Phi29 DNA PolymeraseReaction Buffer (1×) contains 50 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mMMgCl₂, and 4 mM Dithiotheitrol. Buffering reagents interfere with pHmeasurement when using NanoBridges or ISFETs as sensors, and the highion concentrations create a high background level that may interferewith measurements when using the NanoNeedle sensor.

Thus in some embodiments it is desirable to use buffer reagents withlower concentrations of pH buffers and or lower total ionconcentrations.

In some embodiments, it is desirable to use very low ionic strengthreagents, in order to maximize the Debye length. For such embodiments,it may be desirable to use reagents that have no more salt than isneeded for the enzymatic reaction. For such reagents, it is desirable tominimize the amount of salt, for example reducing or minimizing theamount of NaCl or KCl, and using sufficient Mg. Sufficient Mg mayinclude a concentration equal to the concentration of nucleotides usedin the reagent, with additional Mg to act as counter ions for the DNA,and additional Mg for polymerase in the flow cell associated with theDNA. Thus the concentration needed will be a function of nucleotideconcentration, the amount and length of DNA in the flow cell, the numberof polymerase molecules, and the volume of reagent used.

In some embodiments where the ionic concentration is very low, the pHmay be influenced by the surrounding air. Any residual CO₂ which mayremain after any efforts to minimize the presence of CO₂ formingcarbonic acid, reduces the pH. Buffering reagents contribute to theionic concentration; so minimizing the amount of buffering is alsodesired. Mitigating conflicting needs between having sufficientbuffering and having sufficiently low ionic strength may be accomplishedby several embodiments. One embodiment uses two buffers together, forexample, combined Tris and HEPES as opposed to Tris HCl, whereby bothTris and HEPES can contribute to buffering. Ideally both buffers wouldhave a high molecular weight/charge for reduced mobility. In anotherembodiment, organic reagents which may be miscible with water may beused, such as an alcohol (e.g., ethanol).

In some embodiments it is desirable to eliminate any monovalent cationssuch as Na⁺ or K⁺ from the buffer to avoid competitive reactionsrelative to divalent Mg++ causing changes to the counterion distributionon the DNA or beads.

In some embodiments, the charge associated with a bead may diminish therange of an electrical charge/conductivity sensor, or degrade the signalto noise of the sensor. As a result, in some embodiments, it is desiredto minimize the amount of charge present on the surface of the bead, byfor instance changing the amount of sulfate or other negative chargedmoieties on the surface. In some embodiments, it may be desired to havea small amount of negative charge, so that DNA or nucleotides do notbind to the surface of the beads, but not sufficient charge so thatthere is not significant reduction in the dynamic range of the sensor.In other embodiments the beads may have a slight positive charge suchthat when DNA primers are attached the beads become negatively charged.Solutions containing solvents such as ethanol can be used to solvate thebead to allow attachment of the DNA.

In a yet further embodiment, ligation may be used rather thanpolymerization. Four pools of probe oligos may be used, where the firstbase of each probe in a single probe pool is the same. The probes mayuse a reversibly terminated tail, or may have a native tail, such thatmultiple ligations may occur, with concomitant increases in signallevels. In a manner similar to the use of multiple dNTPs and polymerase,more than one pool of oligos (with all probes starting with a singlebase) may be combined, again with concomitant increase in the number ofligations and signal levels. The second strand may be removed and a newprimer introduced wherein the length of said primer may be shorter orlonger than the length of the previous primers.

In yet another embodiment, the attached DNA molecule may have a hairpinprimer, where a portion of the hairpin primer has a restriction site.Subsequently, after completion of the primer extension and associateddetermination of the sample DNA sequence, the restriction site may becleaved by an appropriate endonuclease enzyme or nicking enzyme, and theextended primer may be melted off by changing one of the temperature orpH of the solution in which the sample DNA is solvated. The sample maythen be re-sequenced after restoring the temperature or pH of thesolution in which the sample DNA is solvated to the conditionsappropriate for primer extension, including appropriate concentrationsof nucleotides and cations. In an alternative embodiment, a stranddisplacing enzyme, or an enzyme with 5′ to 3′ exonuclease activity maybe used, obviating the need to remove the second strand.

In a further embodiment, a linkage may be provided which may bechemically cleaved, obviating the need for enzymatic cleavage.

In some embodiments, it is desirable to minimize the number of counterions associated with the polymerase and or any other helper proteins.Thus it may be desirable to substitute charged amino acids in thepolymerase such as Glu, Asp, Lys, His, and Arg with very conservativesubstitution such as respectively Glu to Gln, Glu to His, Asp to Asn,Arg to Gln, His to Tyr, Lys to Arg, Lys to Gln, Lys to Glu, or with aconservative substitution such as Glu to Arg, Glu to Asn, Glu to His,Glu to Ser, Asp to Gln, Asp to Ser, Arg to Asn, Arg to Glu, Arg to His,His to Arg, His to Gln, His to Glu, lys to Asn, Lys to Ser, using themore divergent BLOSUM45 alignment. Said substitutions may be from acharged amino acid to an uncharged amino acid, or may be from anuncharged amino acid to a charged amino acid, where the amino acid whichis changed from an uncharged amino acid to a charged amino acid may beadjacent to a charged amino acid of the opposite charge, whereby thecharge may be shared between said charged amino acids, obviating orreducing the need for a counter ion.

In some embodiments it may be desirable to make said substitutions onportions of the protein which interact directly with the fluidicenvironment, as opposed to interacting with ssDNA to which said proteinmay be bound. In some embodiments it may be desirable to add additionalpositively charged amino acids in locations which interact and bind withsaid ssDNA in order to provide tighter binding.

For example as seen in FIG. 10 (Hollis et al PNAS 98 17 9557), whichshows in darker grey as part of 1002 the positively charged portions ofa single SSB (normally ternary in vivo), which thereby shows theportions of said SSB which bind ssDNA. FIG. 10 conversely shows indarker grey as part of 1000 the portions of said SSB which may bepositively charged, and which may therefore cause additional counterions to accumulate as the SSB binds to the ssDNA, causing an increasedbackground current change due to either the influence of charge on thesensitive area of a NanoBridge, or by locally increasing theconductivity of the volume proximate the ssDNA. In some embodiments, aDNA binding protein, which may be a polymerase, or a protein with ssDNAor dsDNA binding affinity, may be mutated such that the binding of saidprotein(s) to a DNA strand(s) of interest results in a lower backgroundcurrent change than might occur if a native DNA binding protein(s) wereto bind to said DNA strand(s) of interest. In some embodiments, thebackground current change occurs as a result of the binding of a mutatedprotein(s) to said DNA strand(s) of interest may result in no observablechange in the background current relative to the background currentwithout a protein(s) being bound to said DNA strand(s) of interest as aresult of the reduced charge of the mutated protein which interacts withthe fluid, or as a result of the mutated protein(s) reducing the numberof counter ions which may be associated with said DNA strand(s) ofinterest as a result of increased charge interaction between saidmutated protein(s) and said DNA strand(s) of interest. In otherembodiments, the background current change occurs as a result of thebinding of a mutated protein(s) to said DNA strand(s) of interest mayresult in a decrease in the background current relative to thebackground current without a protein(s) being bound to said DNAstrand(s) of interest as a result of the reduced charge of the mutatedprotein which interacts with the fluid, or as a result of the mutatedprotein(s) reducing the number of counter ions which may be associatedwith said DNA strand(s) of interest as a result of increased chargeinteraction between said mutated protein(s) and said DNA strand(s) ofinterest.

In other embodiments, a primer may be provided which has a nick site. Instill further embodiments, multiple adjacent primers may be provided,obviating the need for a nicking endonuclease. The primers may becomplementary to a ligated primer, or may be complementary to a targetedsection of DNA. The sequencing primers may comprise all or part ofprimers used for clonal generation via an amplification reaction, or maycomprise regions which may be not used as part of the primers foramplification, or may comprise both regions used for primers in anamplification reaction, and a region which is not used for amplificationreaction.

In some embodiments, the quality of measurements the signal may bedependent on the sharpness of the reagent front hitting the sensor, andthe collection time window size may be dependent on when the reagentcontacts the sensor, particularly for detecting a transient pH change asa result of a base incorporation. The sharpness of the wavefront may bereduced, by for example, diffusion while the reagent is moved throughfluidic lines, valves and the flow cell itself. To better control thetiming and to minimize the diffusion, particularly to minimize the timeneeded to effectualize a reagent delivery an electric field repulsivecharge may be used to hold the active reagents, for example the dNTPsmay be held away from the sensor in the axis perpendicular to the planeof the sensor array during the initial delivery along the length of theflow cell from the input port to the output port. Later the electricfield can be turned off or reversed to draw the reagents to the sensor.In some embodiments the electric field can be activated on differentsections of the sensor array at different times to allow better controlof the readout time window.

In some embodiments, the flow of reagents through the flow cell may belaminar, and reagent(s) delivered to/on the top of the reaction chamberwill stay there with mixing occurring only due to diffusion in the axisperpendicular to the flow of the reagent. This may be used to retardincorporation until desired. In some embodiments both laminar andelectric fields could be combined to control the delivery to the sensor.

In other embodiments the temperature can be kept low, such thatpolymerase activity is minimized while the reagents are delivered andthen brought up to allow the reaction to start.

To minimize diffusion effects, all nucleotides needed for sequenceanalysis may be present in the system, and the only reaction triggeringfactor may be magnesium ions. Magnesium ions have a higher diffusionrate than other components of the reaction such as polymerase and dNTPs.In these or other embodiments, the temperature of the reservoir andreaction may be kept below the activation temperature of the polymeraseenzyme. Reactions can thereby be triggered by precise temperaturecontrol, thereby overcoming diffusion limitations. For example, in theseembodiments the parallel reactions of the array may be almostsimultaneous. Activation temperatures are known in the art and/or may bedetermined experimentally for any particular embodiment. The activationtemperature for Klenow polymerase is approximately 4° C., and theactivation temperature for Taq is approximately 60° C. In still otherembodiments, the reaction may be triggered by introduction of a requiredreaction co-factor, which may be sequestered in nanoparticles orvesicles prior to the reaction, and released with the appropriateexternal stimulus (e.g., laser or temperature).

In some embodiments, as a part of the sample preparation process,“barcodes” may be associated with each sample. In this process, shortoligos are added to primers, where each different sample uses adifferent oligo in addition to primer. The primers and barcodes areligated to each sample as part of the library generation process. Thusduring the amplification process associated with generating each colony,the primer and the short oligo are also amplified. As the association ofthe barcode is done as part of the library preparation process, it ispossible to use more than one library, and thus more than one sample, ingenerating the clonal populations. Synthetic DNA barcodes may beincluded as part of the primer, where a different synthetic DNA barcodemay be used for each library. In some embodiments, different librariesmay be mixed as they are introduced to a flow cell, and the identity ofeach sample may be determined as part of the sequencing process.

Sample separation methods can be used in conjunction with sampleidentifiers. For example a chip could have 4 separate channels and use 4different barcodes to allow the simultaneous running of 16 differentsamples. This permits the use of shorter barcodes while still providingunambiguous sample identification.

Nanosensors and Detection Methods and Systems

In some embodiments, a charge or pH sensitive detector is used todetermine the sequence of a DNA colony. A colony may be generated on abead, the bead may be transferred to a sensor location, provided withprimers, polymerase, and dNTPs while observing the change in charge orpH due to the incorporation of dNTPs. There may be a one to onecorrespondence between a sensor location and a colony.

In embodiments of the devices disclosed herein, a plurality ofnanoneedle sensors are employed having at least one electrode formed inthe shape of an arc conforming to the edge of a depression where one ofthe plurality of magnetic beads sits.

The nanosensor is a sensor designed to detect beads or particles lessthan one of 0.1, 1, 5, 10 or 20 micrometers as measured on the diameteror the long axis for non-spherical beads or particles. Alternatively,the sensor may be sensitive to moieties associated with said beads orparticles, or with reaction products or byproducts wherein the reactionincludes a moiety associated with said bead or particle. Said moietiesmay include DNA fragments, hydrogen ions, or other ions which may becounter ions and thus associated with said beads or particles ormoieties bound or associated with said beads or particles. Nanosensorscan include NanoBridge, NanoNeedle or ISFET sensors. A NanoNeedle may bean impedance measuring sensor including two electrodes situated tomeasure the conductivity of the local environment between the activearea of the electrodes. “NanoBridge” refers to a resistive device whichmay include a sensor which may respond to charge proximate to the activearea of said resistive device, and wherein said resistive device mayfurther be a semiconductor device.

In some embodiments, the NanoNeedle functions as a pH sensor, asdescribed in U.S. Provisional Application 61/389,490 titled “Integratedsystem and methods for polynucleotide extraction amplification andsequencing,” which is hereby incorporated by reference in its entirety.

The sensors may be used for detection of transient properties associatedwith incorporation events as described in U.S. Pat. No. 7,932,034, whichis hereby incorporated by reference in its entirety.

In embodiments of the devices disclosed herein, a plurality ofnanobridge sensors are employed having an active area partiallyencircling, and in immediate proximity to, one of the plurality ofmagnetic beads. For example, the radius of the active area maybe lessthan the radius of the magnetic bead.

In embodiments, of the device, the plurality of nanobridge sensors areadapted to measure the incorporation of nucleotides into apolynucleotide, and the nanobridge sensors each have an active area anda conducting element. The conducting element having a work function thatmatches a work function of the active area.

In some embodiments of the current invention, the sensors may beNanoNeedle sensors, nanobridge sensors, ISFET sensors, ChemFETs,nanowire FETs, carbon nanotube FETs, other types of charge,conductivity, or pH detecting sensors, or a combination of differenttypes of sensors at each sensor location wherein a bead or colony may belocated for sequencing reactions. An individual sensor may detect usingonly a single modality such as charge, conductivity, or pH, or anindividual sensor may detect using more than one modality, such asresponding to both charge and pH. The sensors may provide similarinformation, or they provide complementary information. For example, onesensor at a sensor location may respond to changes in pH, while anothersensor at a sensor location may respond to changes in conductivity. Insome embodiments, one sensor may detect local changes in pH,conductivity, or charge, while another sensor is used as a reference. Inone embodiment, the reference sensor may be placed so that it does notcontact any reagents, and may be used to compensate for changes intemperature, power supply voltages, etcetera.

The change in charge concentration may result from other sources,including binding of DNA to DNA attached directly to the sensor, whichmay be either a nanobridge, a NanoNeedle, or a FET, or may result frombinding of charged cDNA, RNA, proteins, lipids, carbohydrates; thechange in charge may also result from an enzymatic reaction, or anyother chemical reaction which may be sufficiently localized as tolargely occur within the sensing region of one sensor, and within thesensing region of another sensor.

In some embodiments, a combination of NanoNeedle sensors, nanobridgesensors, and magnetic retention structures are used. The NanoNeedlestructures may be located under the magnetic structures that make up themagnetic array elements, or the NanoNeedle structures may be located ontop of the magnetic structures that make up the magnetic array elements.In other embodiments, the NanoNeedles may be located orthogonally, or atsome other angle with respect to the structures that make up themagnetic array elements.

In some embodiments the NanoNeedle sensor may measure the impedancechange due to ions generated by a DNA polymerization event.

In other embodiments, the NanoNeedle measures the impedance surfacechange due to the incorporation of the DNA. Each base of DNA has anegative charge. As bases are added the charge becomes more negative.This additional charge attracts positive counter-ions which can changethe conductivity of the surface of the DNA coated bead. This impedancechange may also result from molecules that bind to DNA.

Because the charge is associated with a fixed molecule (DNA bound tobead) the local fluid environment is changed from the polymerizationcondition. For example, one buffer can be used for the baseincorporation and a second buffer can be used to measure theconductivity change from a previous measurement.

In some embodiments a NanoNeedle sensor is configured to measure theimpedance change of a bead as bases are added to the template DNAattached to said bead. In other embodiments, said DNA template may beattached or associated with the substrate or a coating on saidsubstrate, or may be attached or associated with the device electrodesor coatings on said electrodes. To improve performance it is desirableto reduce the other impedances.

The sensor impedance may be dominated by other impedances. For example,the impedance of the bulk reagent between the electrodes and the Debyelayer associated with the bead may be large relative to the impedancethrough the Debye layer associated with the bead if the physicalalignment is not good between the electrodes of a NanoNeedle and a beadto which DNA is bound. If, for example, the impedance of the bulkreagent constitutes 90% of the total impedance between the electrodes,and the impedance of the DNA on the bead and its associated counter ionsconstitutes 10% of the total impedance between the electrodes, a 1%change in the impedance of the DNA and associated counter ions willresult in a 0.1% change in the total impedance between the electrodes.The impedance of the bulk reagent between the electrodes may be smallrelative to the impedance through the Debye layer associated with thebead if the ion concentration of the bulk solution is high.

FIG. 16 schematically illustrates a simplified circuit 1600 for aNanoNeedle sensor with bead. The sensor may have parasitic capacitance1614 and parasitic resistance (not shown). The sensor may further havedouble layer capacitances 1610 associated with each electrode 1612. Theresistance resulting from the counter ions associated with the DNA orother sample bound, attached or associated with the bead 1606, and maybe in parallel with the resistance resulting from the bulk fluid 1602,the resistance from conductivity through the counter ions associatedwith charge on the surface of the bead 1606, and the resistance fromconductivity through the counter ions associated with charge on thesurface of the sensor 1608. Additional portions of the circuit that mayadd complexity include resistances (not shown) between the double layercapacitance 1610 and the resistance from conductivity through thecounter ions associated with charge on the surface of the bead 1606, andthe resistance from conductivity through the counter ions associatedwith charge on the surface of the sensor 1608.

Thus in some embodiments, it is desirable to minimize the distancebetween both electrodes and the bead. In other embodiments, it isdesirable to measure the counter ions from as much of the bead aspossible, allowing averaging from as much of the entire surface of saidbead as possible. In some embodiments it may be desirable to positionsaid electrodes at opposite sides of said bead, allowing current to flowover the entire surface of said bead in as even a manner as possible. Inother embodiments, it is desirable to have electrodes that are not assmall as might be possible, so that the current density of the currentpath emitting from said electrodes is not significantly higher than thecurrent path at the point in the current path wherein the currentdensity is smallest. For example, if an electrode could be madeinfinitely small, the current density emanating from said infinitelysmall electrode would be infinitely large. In another example, theelectrodes comprise spherical caps at opposite ends of said bead, andthe circumference of the circle formed by the spherical cap is one halfof the length of the great circle of said bead. The maximum currentdensity will be twice as high at the spherical cap as it is at the greatcircle midway between the spherical caps. In some embodiments the ratioof maximum current to minimum current over the surface of the bead maybe 2 to 1; in other embodiments, the ratio of maximum current to minimumcurrent over the surface of the bead may be between 2 to 1 and 3 to 1,between 3 to 1 and 4 to 1, between 4 to 1 and 6 to 1, between 6 to 1 and9 to 1, between 9 to 1 and 15 to 1, between 15 to 1 and 30 to 1, orbetween 30 to 1 and 100 to 1.

In some embodiments, the electrodes are fabricated with semiconductortechnologies, and the area of the electrode adjacent to the bead is of aheight equal to the thickness of the electrode. It may be desirable tohold the electrode a small distance from the bead, such as from 0.1Debye lengths, to 0.3 Debye lengths, from 0.3 Debye lengths, to 1.0Debye lengths, from 1.0 Debye lengths, to 3.0 Debye lengths, from 3.0Debye lengths, to 10.0 Debye lengths, or from 10.0 Debye lengths, to 100Debye lengths. The Debye length is considered to be an additivecombination of the Debye length of said bead and said electrode.Alternatively, it may be desirable for the electrode to have a lengththat is a fraction of half the circumference of the great circle of thebead. Said fraction may be from 0.01 to 0.03 of half the circumferenceof the great circle of said bead, from 0.03 to 0.1 of half thecircumference of the great circle of said bead, from 0.1 to 0.3 of halfthe circumference of the great circle of said bead, from 0.3 to 0.75 ofhalf the circumference of the great circle of said bead. In someembodiments, the system maintains a distance between the electrode andthe bead, and fabricates the electrode so that it is a fraction of halfthe circumference of the great circle of the bead.

In some embodiments, it may be desirable to reduce the current passingthrough the bulk reagent, in order to maximize the portion of themeasurement current which passes through the counter ions associatedwith DNA bound to, attached to, or associated with a bead. As a result,in some embodiments, it is desirable to physically reduce the volume ofbulk reagent in proximity to the bead, such that the impedancecontribution from the DNA counter ions is maximized. In otherembodiments, it is desirable to minimize the surface area of thestructure which retains the bead in proximity to the electrodes formeasurement of the DNA counter ions. In a further embodiment, it isdesirable to minimize the zeta potential of the bead and or surface(s)of the structure which retains the bead in proximity to the electrodesfor measurement of the DNA counter ions.

The NanoNeedle structures may be fabricated in an array of NanoNeedles,permitting large numbers of single DNA molecules or colonies to besequenced at the same time.

As shown in FIG. 17, a NanoNeedle sensor structure 1700 may befabricated with a Silicon substrate 1701, and may have a 800 nm deepchannel 1702 etched in said substrate. A silicon oxide layer of 200 nmthickness 1703 may be fabricated on the substrate, followed by aconductive p+ silicon layer of 80 nm thickness 1704, followed by asilicon oxide layer of 30 nm thickness 1705, followed by a conductive p+silicon layer of 80 nm thickness 1706, followed by a silicon oxide layerof 20 nm thickness 1707. The channel may be created after the structureis fabricated. The structure may be generated such that an oxide layeror a resist layer covers all sections which may be to be retained in thefinal structure. A chemical wet etch, a plasma etch, or a vapor phaseetch may be utilized to remove the silicon or other similar substratefrom under the structure. The conductive tip of the structure may thenbe exposed using an ion milling step.

All of the thicknesses may be varied, as may the materials. The channelin the substrate may alternatively be fabricated using an oxide layer,with a resist layer in the volume of the channel. The layers of Oxideand conductors may then be fabricated on top of the oxide and resist,obviating the need to under-etch the structure. FIG. 18 illustrates asingle ended NanoNeedle array fabricated in a manner similar to thatschematically depicted in FIG. 17.

As shown in FIG. 19 such a structure may have sensors 1901 on both sidesof a channel 1902 formed in a substrate 1903. Polymerase and or targetDNA 1904 may be attached to the active area of the sensor. The sensoritself may be used to electrophoretically and or dielectrophoreticallylocalize the polymerase and or target DNA to the active area of thesensor. The target DNA may be a single double stranded, single strandedDNA target, or a circularized DNA target, or a local amplification maybe done in place on the active area of the sensor, as described inPCT/US2011/054769. FIG. 20 illustrates an interdigitated NanoNeedlearray fabricated in a manner similar to that schematically depicted inFIG. 19.

Nucleotides or probes 1905 may be then be provided, and a sequencing bysynthesis process, or a sequencing by ligation process may commence.

To improve the sensitivity of either the NanoNeedle or the NanoBridge, alocal amplifier may be provided. The amplifier may be either a BJT or anFET. In some embodiments, an amplifier is used with one amplifiercircuit for each sensor or with multiple sensors sharing the sameamplifier. In other embodiments, some amplification may be associatedwith each sensor, and additional amplification and or other associatedcircuitry is shared or multiplexed between different sensors. The sensorcan be fabricated as a narrow structure, and can be etched under thestructure so that both sides may be accessible to changes in pH, or tochanges in conductivity. The surface of the device may be rough,permitting greater surface area for binding of sample molecules. Thesurface associated with the electrodes of a NanoNeedle may be gold orplatinum, or may be platinum black, iridium oxide, or Ppy/PSS toincrease the surface area and the associated double layer capacitance.

Electric concentration of ions may be effected, concentrating the DNA,polymerase, primers nucleotides and other reagents as needed to theactive area of the NanoNeedle or NanoBridge sensor. Said concentrationallows much more of the sample to be attached or associated with eachsensor, mitigating the need for whole genome amplification.

Another factor which may prevent optimal measurement of the impedance ofDNA on the bead includes counter ions which result from the Debye layerassociated with the zeta potential of the surface of the bead and or thecounter ions which result from the Debye layer associated with the zetapotential of the surface of the sensor. These counter ions may result ina current which may be in series and or in parallel with the desiredcurrent associated with counter ion of the DNA on the bead. Further, asthe zeta potential varies, the Debye length and the number of associatedcounter ions may vary concordantly. Said zeta potential may vary withchanges in buffer conditions including changes in pH, salt concentrationand various other factors. Said concordant variation may thus confusemeasurements of the DNA. Thus it is desirable to both minimize the zetapotential, and to minimize variations of zeta potential with variationsin buffer conditions.

In some embodiments, the sensor may be fabricated with silicon. Silicondioxide has a significant zeta potential magnitude at pHs typicallyuseful for polymerization activity, such as pH 7 to 9; but the zetapotential magnitude of silicon nitride is significantly less than thatof silicon dioxide. Thus in some embodiments, it silicon nitride is usedat the interface between the silicon sensor device and any componentswhich may come into contact with the silicon sensor device, to therebyminimize the zeta potential and concomitant current through counter ionswhich may be in the associated Debye layer.

In some embodiments, a coating is applied over the surface of thesensor. The sensor may be fabricated of silicon, silicon dioxide, PDMS,Topaz™ or other various polymers or combinations thereof, where saidelectrode and or coating may comprise materials such as TiO², ZrO², orIndium Tin Oxide, BaTiO³, such that the zeta potential and the resultingDebye layer are significantly reduced. In other embodiments, surfacecoating(s) such as PEG (Poly Ethylene Glycol), PTFE, poly L lycine,acrylate, methyl cellulose, n-dodecyl-B-D-maltoside, acrylamide,fluorinated alkane chains, or other cross-linked or partiallycross-linked polymers are incorporated to modify the zeta potential, orcombinations of surface coatings are used to similarly minimize the zetapotential and concomitant Debye length. In other embodiments the zetapotential magnitude is reduced by protecting the silanol groups with acompound such as trimethylchlorosilane which decreases the number ofionizable silanol groups.

In some embodiments, it is desirable to reduce the zeta potential of abead on which DNA to be sensed is attached, thereby reducing theconcomitant current resulting from counter ions associated with thesurface of the bead due to said zeta potential. Thus in some embodimentsit is desirable to fabricate the bead of a material with a low zetapotential at the pH levels anticipated for effective polymerization, orthe bead may be coated with a material with a low zeta potential at thepH levels anticipated for effective polymerization, such as such as PEG(Poly Ethylene Glycol), PTFE, poly L lysine, acrylate, methyl cellulose,n-dodecyl-B-D-maltoside, acrylamide, fluorinated alkane chains, or othercross-linked or partially cross-linked polymers are used to modify thezeta potential, or combinations of surface coatings may be used tosimilarly minimize the zeta potential and concomitant Debye length.

In some embodiments, it is desirable to minimize variations in pH whichmay result from buffer reagents, while it may be simultaneouslydesirable to minimize ionic concentration. As a result, it is desirableto use reagents with little buffering capacity while maintaining a fixedpH. Buffers may in some cases be degassed as part of an assay or method;said buffers may then be subject to changes in pH as CO₂ dissolves intothe buffer reagent. In some embodiments, it may be desirable to restrictthe interaction between CO₂ and buffer reagents. Thus it may bedesirable to exclude atmospheric gases, and to provide other gases whichdo not include CO₂ such as Nitrogen, Argon, or other purified gases, ormixtures of gases which do not contain CO₂ or other gases which mightotherwise dissolve into said reagent buffer, and thus change the ionconcentration and or pH.

In some embodiments, the system includes an external gas source such asan industrial gas cylinder. In some embodiments said gas cylinder isexternal to an instrument where the fluidics resides. In otherembodiments, the industrial gas cylinder is placed within a compartmentwithin the instrument. In other embodiments, a CO²scrubber/degasser/debubbler is used such as a regenerable metal oxidesystem, a Kraft process system, an activated carbon system, a membranesystem which may use a membrane such as the Systec AF® or Poridex™. SaidCO₂ scrubber/degasser/debubbler may be built within said instrument, ormay be external to said instrument.

In some embodiments, it may be desirable to bring the sensor electrodesfor a sensor such as a NanoNeedle sensor into close proximity to a bead,in order to minimize the amount of bulk reagent volume between theNanoNeedle electrodes and the bead. One embodiment may have a bead heldin a depression as shown in FIGS. 2A, 2B and 2C. The depression may beformed of a material which is deposited on a substrate, and the materialforming the depression may have a pair of NanoNeedle electrodes formedupon said material. The electrodes may be formed in an arc conforming tothe edge of the depression, and thus to the edge of the bead. In someembodiments said depression may be fully accessible to fluids on oneside, or two sides, or said depression has a width to depth ration ofless than 1.0, or may have a width to depth ratio of 1.0 to 0.9, 0.9 to0.8, 0.8 to 0.7, 0.7 to 0.6, 0.6 to 0.5, 0.5 to 0.4, 0.4 to 0.3, 0.3 to0.2, 0.2 to 0.1 or 0.1 to 0.01.

In some embodiments, the depression minimizes the volume of reagent inproximity to the bead. The depression may be shaped so as to conform tothe shape of the bead, whereby the bottom of the depression is narrowerthan the cross section of the depression at the height of theelectrodes. In other embodiments, the electrodes are covered by a layerof additional material, such that the effective depth of the depressionis greater than half the diameter of the bead, further reducing thevolume of bulk reagent proximate the bead.

The electrodes may thus be touching the surface of the bead, or may bewithin the Debye length of the surface of the bead or particle and theDNA attached or bound thereto. In some embodiments, the electrodes arecurved such that the electrodes follow a curve with a radius similar tothe radius of the bead, permitting better coupling between the electrodeand the bead. Such a device permits a minimum influence on the totalimpedance between the NanoNeedle electrodes by the bulk reagentsolution, and a maximum influence by the DNA attached to or bound to thesurface of the bead or particle or the counter ions near the DNA.

In some embodiments, a NanoNeedle has the active area of the sensorshaped to fit a bead or other sample retaining mechanism. It may, forexample be shaped in an arc, with the curve of the arc oriented so as toalign with the curve of a bead. It may also have one NanoNeedle of aNanoNeedle pair configured such that it is offset or “shorter” than theother NanoNeedle of said NanoNeedle pair, such that the inner radius ofthe arc has a larger diameter, and the same centroid. Said offset maypermit an increase in the volume of the sensing region associated withthe NanoNeedle pair, and may further change the orientation of the fieldassociated with the sensing region and thus orientation of the sensingregion, so that the sensing region is more oriented towards the centerof the bead, rather than parallel to substrate.

In an alternative embodiment as shown in schematic side view FIG. 21Aand schematic top view FIG. 21B, one electrode 2105 may be attacheddirectly to the substrate 2102 or on another layer upon said substrate,allowing isolation from said substrate. The second electrode 2105 fn theNanoNeedle may be attached upon a dielectric 2113 portion of the sensorwhich is utilized to position the bead or particle 2101 in a fixedlocation. The bead or particle 2101 is thus in contact with bothelectrodes 2101, 2105, minimizing the influence of the bulk reagentsolution on the total impedance between the NanoNeedle electrodes, asopposed to the impedance resulting from the counter ions within theDebye length associated with the bead or particle and the DNA which isattached or bound to the bead or particle.

In some embodiments, one or both electrodes may be fabricated such thatsaid electrodes conform to the curve of the bead so as to provide alower and more regular impedance between the electrode(s) and the bead.The curvature may abut the edge of a depression, or may be slightlyfarther from the edge of the depression so as to allow a larger area ofinterface and a lower current density.

In a further embodiment as shown in FIG. 21C, the bead or particle 2101may be held in place on a substrate 2102. A first electrode 2105A of aNanoNeedle 2100 may be attached directly to the substrate 2102, or to anadhesion layer (not shown) adhered to said substrate 2102. A dielectriclayer 2114 may then be fabricated so as to cover said first electrode2105A. A second electrode 2105B of a NanoNeedle 2100 may then befabricated above the dielectric 2114 and said first electrode 2105A ofthe NanoNeedle 2100. The second electrode 2105B may be shorter, so as toconform to the curve of the bead or particle. The difference in thelength will be a function of the diameter of the bead or particle 2101,and the thickness of the two electrodes 2105 and the dielectric 2114between the electrodes 2105. In this manner the electrodes 2105 may bein contact with the bead or particle 2101, or may be in very closeproximity to the bead or particle 2101, such that the impedanceresulting from the counter ions within the Debye length associated withthe bead or particle 2101 and the DNA which is attached or bound to thebead or particle 2101 is greater than the impedance of the bulk reagent.

In a further embodiment, the electrodes are fabricated such that saidelectrodes do not abut the edge of a depression, but may be insteadfabricated a short distance from the edge such that the current densityin immediate proximity to the electrode may be reduced.

In FIG. 22A, a NanoNeedle 2200 is schematically illustrated in a sideview wherein said NanoNeedle 2200 has electrodes 2205 on each side of adepression in a dielectric 2203, wherein a bead 2201 may be retained ona substrate 2202, and metalization 2204 to said electrodes 2205 may beused. As shown in FIG. 22A, the dielectric material 2203 may be similarin thickness to one half the diameter of the bead 2201, and thedepression width may be slightly larger than the diameter of the bead2201 while still allowing the bead 2201 to be within the Debye length ofsaid bead 2201 with respect to both electrodes 2205. Said thickness ofthe dielectric 2203 may of a thickness which permits retention of thebead 2201, and maintains said bead 2201 within a Debye length of saidbead 2201 of both electrodes 2205.

In FIG. 22B, a NanoNeedle 2200 is schematically illustrated in a sideview where the NanoNeedle 2200 has electrodes 2205 on each side of adepression in a dielectric 2203, where a bead 2201 is retained above asubstrate 2202, and metalization 2204 to the electrodes 2205 is used. Asshown in FIG. 22A, the dielectric material 2203 may be less than onehalf the diameter of the bead 2201, potentially the dielectric material2203 thickness is one quarter to one third the diameter of the bead, andthe depression width may be less than the diameter of the bead 2201 suchthat the bead 2201 is suspended above the substrate 2202. The closeproximity of the bead 2202 and the electrodes 2205 maintains a spatialproximity between the bead 2202 and the electrodes 2205 such that thebead 2201 is within a Debye length of the bead 2201 of both electrodes2205.

In FIG. 22C, a NanoNeedle 2200 is schematically illustrated in a topview wherein the NanoNeedle 2200 has electrodes 2205, which are curvedto maintain close proximity to the bead as a result of the depression inthe dielectric material 2203 being smaller in diameter than the diameterof the bead 2201, such that the bead is held in immediate proximity overan arc corresponding to the point of contact or close proximity betweenthe bead 2201 and the electrodes 2205.

FIG. 22D is a three transparent dimensional drawing of a NanoNeedlestructure 2200 similar to that of FIG. 22B and FIG. 22C, but withsubstantial fluidic access to the bead 2201. The bead 2201 is heldsuspended above the substrate 2202, and is instead held againstelectrodes 2205A, which are fabricated above the dielectric material2203, where the electrodes 2205A and dielectric material 2203 are shapedto be within a Debye length of the bead 2201 Debye length, but is notcurved to match the curvature of the bead 2201, such that instead ofhaving a line contact between the bead and the electrode 2205 and ordielectric material 2203, there are three or four point contacts betweenthe bead 2201 and the electrode or dielectric material 2203. FIG. 23Dalso depicts magnets 2208 which apply the force to retain the bead 2201in place in the NanoNeedle structure 2200.

A NanoNeedle may be configured to be in a double spiral or serpentinepattern, in order to increase the length, and simultaneously decreasethe width of the nanobridge channel. A sensing region that is too widewill have a comparatively low impedance, and may have areas of thesensing region which have smaller changes in local charge density thanother regions, for example, at the edges of a bead in comparison withthe center of a bead. The sensing region which is “too wide” may thusalso have smaller changes in impedance, as only a portion of the sensingregion may be significantly affected by a binding or reaction whichresults in a local change in charge. In contrast, a NanoNeedle that istoo long and thin may have an impedance that is so large that anycurrent change may be too small to sense with good signal to noise. Thusthe width and length of the channel associated with a nanobridge sensorwill need to be tuned for the specific application for which saidnanobridge sensor is intended.

In some embodiments, a NanoNeedle is configured to have several activeregions as part of a single NanoNeedle. The active regions are locatedat various locations with respect to a single sample, providing anaverage of several different areas from the sample region, such thatvariations in locations of a sample region for example, slightmisalignment of a bead relative to a sensor or variations in loadingdensities on a surface, will have less affect on the signal to noise fora sensor.

Streaming potential was originally observed by Quinke in 1859, and is awell known phenomenon in capillaries; it is a function of the flow rate,the zeta potential, and the conductivity of the fluid, among otherfactors. Thus a voltage may be impressed on a NanoNeedle, and variationsin the flow rate or distances between electrodes may result invariations either spatially or temporally in the bias impressed on aNanoBridge.

In other embodiments it is desirable to orient NanoNeedle electrodesparallel to the flow of the fluid, so that there will not be apotentially variable streaming potential impressed between theelectrodes of the NanoNeedle, as would be the case if the electrodeswere orthogonal to the flow of said fluid.

In some embodiments, the NanoNeedle is coupled with a local capacitor,or capacitor, associated with one or both electrodes, in order toprevent influence from DC bias levels from the driver circuit or leakagefrom within the chip sensor from influencing the output signal.

In a further alternative embodiment, a Nanobridge sensor structure 2300as shown in FIG. 23 is used instead of the aforementioned NanoNeedlesensor. The Nanobridge sensor may be used in the same manner as theNanoNeedle, including with circularized or linear DNA, a linear orhairpin primer, a polymerase as described for the nanobridge, and mayfabricated as an array.

The NanoBridge sensor structure 2300 may comprise a silicon-on-insulatordevice, comprising a substrate 2360, a dielectric insulator 2310, twohigher doped semiconductor regions 2304A and 2304B, a lower dopedsemiconductor active area region 2305, a further metalization layer 2340which may cover said semiconductor active area region 2305, and whichmay have a further dielectric coating 2350 over said semiconductoractive area region 2305.

In some embodiments, the nanobridge senses local changes in charge.Changes in surface charge of a surface of the nanobridge abutting theflow cell may result from changes in charge in the second layer in theflow cell. These changes in charge on the surface of the nanobridge maythen change the charge distribution in the nanobridge, and thus changethe conductivity of the nanobridge. The area of the nanobridge surfacewhich has charge changes may thus have changes in conductance in theassociated volumes of the nanobridge, while other surface areas of thenanobridge may not have changes in surface charge, and thus may not havechanges in conductance in the associated volumes thereof. Depending onthe type of semiconductor material the nanobridge is constructed of (nor p type), the amount and uniformity of doping in the nanobridgesemiconductor material, and the sign of the charges (positive ornegative) on the surface of the nanobridge, and whether the change incharge is an increase or decrease in the amount or density of surfacecharge may increase or decrease the conductance.

In some embodiments, a change in the charge on a bead located within theDebye length causes a corresponding change quantity or concentration ofcharge locally present in both of the layers of the double layer. Saidchange in the quantity of charge relates directly to the local ionconcentration, and thus also to the surface layer capacitance, and theconductance of the reagent within the Debye length. Said change in thecharge may be either an increase, or a decrease depending on therelative charge of the surface layer and the charge change on a bead.

In some embodiments for sequencing of a clonal bead, a Nanobridge sensoris used. The Nanobridge sensor may be used in a similar fashion to theNanoNeedle.

In an alternative embodiment, the nanobridge detects a local temperaturechange, and thus acts in part as a temperature sensor.

In an alternative embodiment the Nanobridge can be configured to operateas a temperature sensor and/or a pH sensor to detect nucleotideincorporations. This method is further described in US patentapplication 20080166727 titled “Heat and pH measurement for sequencingof DNA,” which is hereby incorporated in its entirety.

The present invention provides methods and systems for polynucleotidesequencing based upon pH and/or temperature detection. In someembodiments, the system and method may further employ (or alternativelyemploy) dyes or quantum dots that allow visual or optical detection ofpH and/or temperature changes. This monitoring may allow monitoring ofthe bulk solution, or may allow local monitoring of the volumeassociated with each colony, or may allow for monitoring of both thebulk solution and the volume associated with each colony.

In other embodiments, an array of NanoBridge sensors is etchedunderneath, so as to further minimize the channel size, and to maximizethe surface area which interacts with the charge resulting from the DNAsequencing reaction. In other embodiments, the array of NanoBridgesensors may not be etched underneath, or may be partially etched so asto provide a more robust structure. In yet further embodiments, thearray of NanoBridge sensors is configured such that it is arranged in acomb configuration, with sensors interleaved between each other fromboth sides, with potential features, such as a potential amplifierarranged alternatively on one side, and then the other. In anotherembodiment, the array of Nanobridge sensors is arranged such thatpotential features, such as an amplifier, are all arranged on one sideof the sensor array.

In some embodiments, a nanobridge sensor is configured such that thewidth and length of the sensing channel is aligned for optimalsensitivity for a sensing application. Variations may relate to thespacing and size of a sample region, the charge associated with sensingthe desired moiety, and the impedance of the nanobridge in nonsensingregions, such as conductive portions of the nanobridge between thesensing region and a local amplifier, or other associated impedances.

In some embodiments, the sensor is a NanoBridge sensor 2400 where theactive area is fabricated such that the active area partially encirclesthe bead or particle 2401, and is in immediate proximity to the bead orparticle 2401, as shown in FIG. 24A, FIG. 24B, and FIG. 24C. The sensormay comprise a substrate 2402, on which a layer of dielectric and orsemiconducting material 2403 may be applied. The active area of theNanoBridge 2405 may be fabricated such that it largely encircles thebead or particle 2401. Metalization lines 2404 may connect to morehighly doped regions of semiconducting material 2404A which theninterface with the active area 2405 of the NanoBridge. FIG. 24A is aside view of a “ring” NanoBridge, where the inner portion of the activearea 2405 is within the Debye length of the bead or particle and the DNAwhich may be bound thereto. The active area may be partly or entirelywithin the Debye length of the bead or particle, resulting in impedanceof the entire active area changing in response to changes in the chargewhich is bound or associated with the bead or particle and/or theincorporation event of a nucleotide or nucleotide analog. The diameterof the ring and the associated supporting structure 2403 may be sizedsuch that a bead fits closely within the ring.

Alternatively, as shown in FIG. 24B, the ring and support structure 2400may be sized to be smaller than the diameter of the bead or particle2401, such that a bead may rest upon the ring of the active area of theNanoBridge 2405, particularly when held by a magnetic field, insuringthat the ring is within the Debye length of the bead or particle 2401and the DNA bound thereto. FIG. 24C is a top view of a NanoBridge 2400implemented with a ring structure, showing the overlap of the bead 2401over the active area of the NanoNeedle 2405, and the electricalconductors 2404 which provide a means to measure the impedance of theactive area 2405.

In some embodiments, the shape of the Nanobridge sensors are optimizedto provide greater interaction with the magnetic or paramagneticparticle. Said Nanobridge sensors may be shaped in a spiral, serpentineor other non linear shape, or a shape that has a variable cross sectionso as to provide more surface area while retaining a narrow channel forcurrent to flow through in the channel of the Nanobridge.

The electrical conductors 2404 may be connected to heavily doped regionsof the NanoBridge (not shown), which then provide electrical connectionto the active area of the NanoBridge 2405. Alternatively, the electricalconductors 2404 of the NanoBridge may be directly connected to theactive area 105 of the NanoBridge with having an Ohmic connection byfabricating the NanoBridge electrical conductors 2404 such that the workfunction matches the work function of the active area of the NanoBridge2405. The value of the work function of aluminum is close to value ofthe work function of lightly doped silicon, but is not a perfect match.To create a more perfect match, an aluminum alloy may alternatively beutilized.

Streaming potential was originally observed by Quinke in 1859, and is awell known phenomenon in capillaries; it is a function of the flow rate,the zeta potential, and the conductivity of the fluid, amongst otherfactors. Thus a voltage may be impressed on a Nanobridge ISFET, or otherchemFET sensors, and variations in the flow rate or distances betweenelectrodes may result in variations either spatially or temporally inthe bias impressed on a NanoBridge.

In some embodiments it may be desirable to use reference electrodeswhich may be fabricated between different NanoBridge or ISFET sensors inan array in order to reduce the variation in the bias voltage impressedon the sensitive area of the sensor. In some embodiments, referenceelectrodes are fabricated in between each NanoBridge or ISFET in anarray NanoBridges or ISFETs on the substrate of the array of saidNanoBridges or ISFETs. Said electrodes may be interconnected bymetalization as part of the fabrication of said Nanobridge or ISFETarray. In other embodiments, sets of said electrodes are interconnectedusing metalization as part of the fabrication of said NanoBridge orISFET array. In other embodiments, the reference electrodes arefabricated such that a fixed or variable number of NanoBridge or ISFETsensors are between each NanoBridge or ISFET in the NanoBridge or ISFETarray. In further embodiments, the bias voltage difference may becompensated for by software or firmware, wherein the effect of thevoltage bias may be measured, mapped and said map is used to compensatefor variations in the signal level from the array of NanoBridges orISFETs.

In some embodiments, a reference electrode is used to bias the reagentfluid relative to the sensor electrodes or active area of the sensordevice, which may be an array of NanoBridges and/or NanoNeedles, orChemFETs. In some embodiments, the reference electrode is configured tobe a part of sensor device. In further embodiments, there are multiplereference electrodes, where one or more of the reference electrodes ispart of or associated with a flow cell associated with the sensordevice. In other embodiments, two or more reference electrodes areassociated with the sensor device. In some embodiments, multiplereference electrodes maintain a substantially similar reagent voltage atall of the members of the array, which might be difficult in a flow cellwhere the fluidic thickness is sufficiently thin as to allow significantresistance over the surface of the sensor array.

In some embodiments, at least one additional electrode may be providedto bias the bulk reagent solution in the flow cell. This electrode couldbe the same electrode(s) used at other times to concentrate sampleand/or other reagents. In some embodiments the voltage impressed on theelectrode(s) may be used to bias the detectors at an optimal point intheir response curve, for example, to provide appropriate offset tooptimize the amount of gain, which may provide maximal signal within theavailable dynamic range of an analog to digital converter, so that A/Dquantization error may be minimized.

In some embodiments, the bias level may be modified as a sequencereaction proceeds, and the amount of charge which is proximate to asensor changes. In some embodiments a reading may be taken using thesensors, after which the bias level may be changed, followed by readingthe sensors again, so that any nonlinearity or unexpected offsets whichresult from changing the bias voltage may be observed and compensatedfor by the software. In some embodiments, positive charge is provided onor near the bead or colony, such that the sensor may be biased to anappropriate level.

In some embodiments, multiple reference electrodes may be used withNanoNeedle sensors, NanoBridge sensors, ISFET sensors, or ChemFETsensors.

Electronic sensors, such as ChemFETs may be designed to have a widedynamic range, as is the case with some pH sensors. They mayalternatively be designed to have a smaller dynamic range, but highersensitivity. In one embodiment, both the dynamic range of the sensor andthe sensitivity of the sensor is optimized, by including an additionalelement to the system which biases the active region. Said element maybe a reference electrode or electrodes, wherein a variable voltage maybe impressed between the reference electrode(s) and the active area ofthe sensor(s) (e.g., ChemFET or NanoBridge). Adjustment of the voltagecan permit highly sensitive detection despite a wide change in theamount of charge interacting with the sensor. For example, a sensor maybe optimized to work with a sequencing reaction where the target DNA is100 base pairs long. Alternatively, if the target DNA is 1000 base pairslong, the sensor may no longer be working within the sensor's dynamicrange. The voltage between the reference electrode(s) and the activearea may then be adjusted so that the sensor is permitted to work withinits dynamic range. If in the course of the sequencing reaction, theextended primer has been extended to 500 base pairs, the sensor mayagain no longer be within its dynamic range. The reference voltage mayagain be modified to bring the sensor within its dynamic range.Additionally or alternatively, a back gate may be used in much the samefashion. In a further improvement, the back gate may be segmented, suchthat there may be different sections of the back gate for differentareas of a sensor array. There may be many sections, so that it ispossible to have an individual back gate for each sensor, permittingcompensation for different sequence dependent rates at which the primeris extended.

In some embodiments, reference voltage(s) are changed when employingNanoNeedle sensors, NanoBridge sensors, ISFET sensors, or ChemFETsensors.

In some embodiments, measurements of polymerase incorporation areperformed to determine the sequence of a DNA target. Multiplemeasurements may typically be needed in order to insure that the profileof incorporation is properly captured and measured, for example todetermine the number of bases which have been incorporated in ahomopolymer run. Such a measurement may measure byproducts of theincorporation reaction, such as PPi or hydronium ions. For a large arrayof sensors, such a measurement may require very high data collectionrates, which may challenge the sensitivity of the sensor, preventinginsufficient signal to noise to provide desired error rates associatedwith the sequencing data. There may be difficulties associated withtrading off the errors associated with phase error, and thus length ofread, and the errors associated with accurately measuring which base,and how many bases have been incorporated. This may be a result ofneeding a low ionic concentration for sensor accuracy, and much higherconcentrations in order for the polymerase to function accuratelywithout phase errors. Thus, in some embodiments, two or more differentreagent conditions are used during sequencing, where at least one set ofreagent conditions is optimized for polymerase accuracy and minimizationof dephasing, and a second reagent is optimized for detection, forexample by having a very low ionic strength. Reading the sensorseparately from the incorporation event may improve the sequencing dataaccuracy and read length. In some embodiments less data is required asthe sensor may no longer be forced to be read at a high data rate tocapture the polymerase incorporation event, but may instead be read asmall number of times, potentially as few as a single time. Theelectronics may also have time constants which may be sufficiently longto permit sensor noise to be significantly reduced. Furthermore, in someembodiments, the reduced data requirements may simplify the dataprocessing hardware, data transfer requirements, and data storagerequirements.

In some embodiments, read buffer may be of lower ionic concentrationthan would be optimal for use for polymerase enzymes. In someembodiments, the ionic concentration of the read buffer may be one thirdthe ionic concentration of the incorporation buffer; or in otherembodiments, the ionic concentration of the read buffer may be one thirdto one tenth the ionic concentration of the incorporation buffer, onetenth to one thirtieth the concentration of the incorporation buffer,one thirtieth to one hundredth the ionic concentration of theincorporation buffer, or one hundredth to one thousandth the ionicconcentration of the ionic concentration of the incorporation buffer.

In some embodiments, the pH of the incorporation buffer and the readbuffer may be substantially the same pH. In other embodiments, the pH ofthe incorporation buffer and the read buffer may be noticeablydifferent, for example, where the pH of the incorporation buffer isoptimized for optimal activity and or accuracy of the polymerase enzyme,such as pH 8.5, while the read buffer is a pH that minimizes theconductivity of the read buffer, such as pH 7.0 (e.g., where theconcentration of OH− and H+ are the same at 10⁻⁷ molar). In someembodiments, the optimal pH for minimal read buffer conductivity isslightly higher than pH 7.0, as the mobility of OH− is lower than thatof H⁺. Thus in some embodiments, the pH of the read buffer is between pH6.5 and pH 8.0, between pH 6.8 and pH 7.5, or between pH 7.0 and pH 7.2,while the pH of the incorporation buffer is between pH 7.5 and pH 9.0,between pH 8.0 and pH 8.8, or between pH 8.3 and pH 8.5.

In some embodiments, different reagent buffers are used with NanoNeedlesensors, NanoBridge sensors, ISFET sensors, or ChemFET sensors.

In some embodiments, an integrator is incorporated with the sensor tomaximize the amount of time given to each sensor in order to reduce theread noise of each sensor. The integrator may include a capacitorassociated with each sensor in the array. In other embodiments, thesensor is configured as a capacitive sensor, where there is no currentflow, but rather an accumulation of charge during a chemistry cycle. Insome embodiments, either an integrating device or a capacitive device,the sensor may have local amplification electronics for each pixel. Inother embodiments, the charge is moved, in a manner similar to a CCD toa readout port.

In some embodiments, integrators are used as a part of the sensor, wherethe sensor comprises NanoNeedle sensors, NanoBridge sensors, ISFETsensors, or ChemFET sensors.

There may be one or more readout ports associated with each device. Insome embodiments, each corner of the device may have a readout port; inother embodiments, there may be many ports along opposite sides of thedevice, allowing a reduced readout rate, and associated improved signalto noise. In other embodiments the readout circuitry can divide thearray into columns or rows. In other embodiments the readout circuitrycan be placed under channel support or channel separation features. Infurther embodiments, there may be multiple sets of readout circuitry,where the array of sensors is divided into multiple subarrays, and themultiple sets of readout circuitry are positioned such that the readoutcircuitry is coincident with the channel support or channel separationfeatures. In some embodiments, it may be desirable to use flow cellswith minimal reagent volume; as such it is desirable to have the heightof the flow cell be as short as possible. For example, it may bedesirable for the flow cell to be 300 microns tall or less, 100 micronstall or less, or 50 microns tall or less. In some embodiments it may bedesirable to use a semiconductor device, which may be one centimetersquare or larger, potentially as large as 10 centimeters square. A flowcell that is wide enough to cover a significant amount of the width ofthe sensor chip may have significant difficulties with mechanicaltolerances due to flatness of one or both major surfaces of the flowcell with respect to the other surface, particularly if one surface is amolded plastic part or a PDMS or similar polymer part. As a result itmay be desirable to use support posts, channels or other support shapesto prevent flatness tolerances from collapsing or expanding beyonddesired tolerances.

In some embodiments, the system may use a sensor, such as a bridgesensor, which is arranged in a manner similar to a Fin FET, whereby twoor three sides of the channel may be accessible to interact with thesurroundings, such as, for example, DNA which is bound to the surface ofthe channel. The sensor channel may have a vertical dimensionperpendicular to the substrate which is greater than the horizontalcross section of the channel. Such a device may have greater sensitivitythan a device which has but a single surface accessible to the sample.

In other embodiments, it may be desirable to use a material to providemore surface area than may be available with a planar or polished planarelectrode. In some embodiments it may be desirable to use blackplatinum, platinum metal sponge, or a platinized metal, which may beplatinized platinum, platinized titanium, platinized irridium,platinized Niobium, platinized tantalum, platinized zirconium, or otherplatinized metals as an electrode material. Said electrode may be areference electrode or may be an electrode as part of a NanoNeedle. Inother embodiments, the electrode surface is fabricated of other membersof the platinum metal group: palladium, rhodium, ruthenium, iridium, orosmium, which may be used in the same manner as platinum to form anelectrode surface with much higher surface area than a planar orpolished electrode would form.

In some embodiments, the process of platinization may include cleaning asupport material, potentially utilizing aqua regia, HCl, and HNO₃,followed by a plating process which may utilize chloroplatinic acid andlead acetate.

In other embodiments, the electrode surfaces may include iridium oxide,titanium nitrate, or polypyrrole/poly(styrenesulphonate) conductingpolymer. Fabrication of said iridium oxide may be effected by sputteringusing standard photolithographic processes. Malleo et al (Review ofScientific Instruments 81, 016104) describe the increase in theeffective interfacial capacitance of different materials relative to abright platinum electrode as ranging from 240 times higher for Platiumblack, 75 times higher for iridium oxide, and 790 times higher forpolypyrrole/poly(styrenesulphonate) conducting polymer.

In some embodiments, sensors with larger surface areas are used withNanoNeedle sensors, NanoBridge sensors, ISFET sensors, or ChemFETsensors.

In some embodiments, a NanoNeedle, NanoBridge, ChemFET or ISFET isfabricated such that the sensor is created on the surface of a substratesuch as silicon, fused silica, glass or other similar material. In otherembodiments, the sensor is fabricated such that it projects verticallyor horizontally above the substrate, such that the sensor is moreaccessible to the fluid and reagents. The greater accessibility to fluidand reagents may decrease the time needed for a sequencing reaction tooccur, allow lower concentrations of reagents to be used, and increasethe sensitivity of the sensor by increasing the surface area associatedwith the active area of the sensor.

In some embodiments, electrode(s) may be fabricated using an angledrotated deposition approach, which may employ glancing angle depositionas described by Zhao et. al. (p59-73 SPIE Vol 5219 Nanotubes andNanowires), or may be fabricated using the PVD methodology described inU.S. Pat. No. 6,046,097, which is hereby included by reference in itsentirety.

FIG. 26 shows data from a one run of a NanoNeedle sequencing reaction,wherein run data is scaled and shown relative to a linear plot of baseincorporations. dNTPs which should not incorporate are shown as mostlyoverlapping previous data, and multiple incorporation base data is shownas having quite good linearity (R2=0.9974).

In alternative embodiments, the system or method detects kinetics ofsingle molecule reactions, such as an enzymatic reaction. In someembodiments, the reaction may a hybridization reaction, whereby a beador particle with a hybridization probe attached thereto may be caused tobe held in place above a sensor, and the change in charge proximate asensor or sensors resultant from a hybridization reaction may bemeasured. In an alternative embodiment, the hybridization probes may beattached on or proximate to the sensors, whereby the change in chargeresultant from the progression of a hybridization reaction may bemeasured. In some embodiments, an electric field may be used toconcentrate DNA from a reagent solution into the volume where thehybridization probes are attached, which may be on a bead or particle,or may be on or proximate to said sensors. Said electric field may be aDC field, an AC field, or a combination thereof.

In some embodiments, a real time PCR reaction is monitored by using thesensor or sensors to monitor the change in conductivity or change incharge present, resulting from the incorporation of dNTPs intoamplicons, and/or the release of pyrophosphate and hydronium ions withhigher mobilities. In an alternative embodiment, an isothermal reactionamplifying target DNA is detected by the resultant change inconductivity from the incorporation of dNTPs into amplicons. In otherembodiments, the sensors monitor the progression of an Immuno-PCRreaction, where a sandwich assay captures an antigen, and the detectorantibody has a probe DNA oligo attached thereto, whereby a realtime PCRassay may then detect and quantify the presence and quantity of antigen,by the detection of the change in conductivity or charge as previouslydescribed. In another embodiment, an isothermal reaction detects andquantifies an antigen of interest.

In some embodiments, protein detection may be effectualized by directmeasurement of the reaction, by measurement of a sandwich assay, or bymeasurement using an aptamer, or by other appropriate methods wherein achange in counter ions or a change in charge associated with the targetwhich may be bound, attached or associated with the sensor.

In another embodiment, a nucleic acid aptamer which is bound to orproximate to the sensor or sensors is used to detect the presence andquantity of a target. The aptamer may bind to the target, changing thecharge which may be detected by said sensor as previously described. Inalternative embodiment, the aptamer is attached on or proximate to saidsensor or sensors, and an increase in the conductivity is detected as aresult of binding of a target thereto.

In a further embodiment, blunt end ligation may be performed withligands that have different binding reagents on the 3′ and 5′ ends ofsaid ligands. The electrodes of the NanoNeedle may be conjugated withthe complementary reagents for binding e.g. the 3′ end of the ligandsmay have a thiol group, and one electrode may be fabricated of gold,while the 5′ end of the ligands may have a PNA sequence, and secondelectrode may have the complement to said PNA sequence. The strand ofDNA may then be electrophoretically and or dielectrophoreticallyconcentrated to the area of the NanoNeedle, where said DNA strand maythen bind with one end associated with one electrode, and the other endassociated with the second electrode of the NanoNeedle.

Polymerase and primer may be bound to the DNA strand, or may beintroduced later. Measurement of incorporation events may then resultfrom direct measurement of the impedance of the DNA combined with themuch larger conductivity of the counter ions associated with the DNA.

In some embodiments, the sensor device, which may be a NanoBridge or aNanoNeedle, generates digital output data. Said digital output maycomprise any of a number of output physical/Data link/protocol formats,including USB2, USB3, Firewire, Gige, single link or dual link DVI,HDMI, S/PIF, ADAT lightpipe, AES3, MADI-X, I²S, AC'97, MC'97, McASP,S-Video, ATM, SONET, SDH, UTP, STP, AUI, HDLC, 802.1, ARP, VLAN, HDLC,ATM, Frame Relay, Q in Q, PPP, BSC, DDCMP, Banyan, CDMA2000, DECnet,CDPD, FUNI, CDMA, X.25, GPRS, GR-303, H.323, NFS, ISDNSS7, TCIP, UMTS,WAP, XNS, MDLP, Infiniband, amongst many others.

Said output may be in compressed format, such as an MPEG 1, MPEG 2,MPEG4, DVA, AVI, MOV, MPG, Video CD, RM, WMA, WMV, WAV, FLC, FLI, BMP,PCX, TGA, TIF, JPG, PCT, GIF, Flash, QuickTime, MP3, or sequencesthereof.

Said sensor device may be configured to have more than one digital I/Oconnection, and may have more than one output format; for example, onedigital connection may be used to control the operation of said sensor,while one or more digital connection sends data from the sensor toadditional device which may be part of an instrument of which the sensoris a part. Said additional device may be a data storage, device, or maybe a computational device. Said additional device may be a GPU, or a setof GPUs such as a GPU array.

Said data may be transferred directly from said sensor to a hard disk,directly from said sensor to a solid state drive, or directly from saidsensor to a GPU, or directly from said sensor to a GPU cluster, GPUblade, or GPU server, a CPU, or the memory associated with a GPU, orCPU. In some embodiments, an instrument or system may have more than onesensor. In such an instrument or system, data may be accumulated frommore than one sensor, and thence sent directly to a hard disk, a solidstate drive, a GPU, a GPU blade, a GPU server, a GPU cluster, a CPU orthe memory associated with a GPU or CPU.

In some embodiments, a single sensor may have more than one digitaloutput. In other embodiments the digital output may be configured todirectly connect to another part of the system, such as a solid statedrive or a memory associated with a GPU or CPU, wherein two or moreparts of the system may be a part of a MCM (Multi Chip Module), or SIP(System in package). Said MCM may be a laminated MCM, a deposited MCM, aceramic substrate MCM or a chip stack MCM. The sensor may be a part ofthe MCM, or may be separate from said MCM. Said sensor may be configuredsuch that said sensor may be removed and a second sensor may beutilized. Said sensor may be interconnected using a socket; said socketmay be a zero insertion force socket for a PGA (Pin Grid Array), a LGA(Lan Grid Array) socket, or a slotket.

The data may be compared with data in a CAM (Content AddressableMemory), or a CAM memory which permits a selectable number of errors inDNA mapping, such as a ternary CAM. Said CAM memory may have multiplelevels in a manner similar to that of TLB (Translation LookasideBuffers), wherein one level of said CAM or TLB may be faster and smallerthan another level of said CAM or TLB.

To improve the sensitivity of either the NanoNeedle or the Nanobridge, alocal amplifier may be provided. The amplifier may be either a BJT or anFET. The sensor can be fabricated as a narrow structure, and can beetched under the structure so that both sides are accessible to changesin pH, conductivity or local charge. The surface of the device may berough, permitting greater surface area for binding of sample molecules.Electric confinement of ions may be effected as will be describedfurther hereafter.

In some embodiments of the current invention, the image sensor array mayuse amplifier designs similar to those in CMOS active pixel imagearrays; these may include three transistor, four transistor, fivetransistor, or six transistor circuits, depending on the signal to noiseneeded, and whether a global shutter equivalent is desired if aintegrating circuit is utilized. Said amplifier structure may bearranged in a one to one correspondence with said image sensor array,potentially providing significantly better signal to noise than mightotherwise be possible utilizing a common amplifier for multiple sensors.

Integrated Systems

The invention further provides methods and systems for localizingsamples and reagents into a volume where a desired reaction or bindingmay occur. The invention is this aspect may eliminate or reduce the needfor whole genome amplification, and thus reduce the coverage needed.

In some embodiments, the DNA sequencer may be part of a larger system,where more portions of the workflow are automated. These portions of theworkflow which may be automated may include cell lysis, DNApurification, DNA amplification, DNA library preparation, colonygeneration, sequencing, primary analysis and base calling, mapping ofsequences to a reference, and determination of whether a genetic diseaseor other genetic characteristic is present. In some embodiments, thesystem may have more functionality, including a means to sort cells,such as cancer cells from blood, utilizing a flow cytometer or affinitypullout of desired or undesired cells.

It may be desirable to process multiple samples in a single chip, sincemany projects do not require the full capacity of a chip. Other projectsmay have a single sample that would exceed the capacity of the chip. Insome embodiments one or more samples could be introduced into theinstrument in individual tubes, tube strips, 96-well plates, 384-wellplates, etc. In some embodiments the sample wells could be sealed toprolong life on the instrument. In other embodiments the plates may becooled to increase sample life. In other embodiments the samples couldbe accessed in a software selectable manner by a robotic pipettor.

Prior to amplification the beads will need to be loaded with a singleDNA fragment in order to create monoclonal beads. Typically the DNAconcentration is determined and then it is introduced to beads in adilute form so that on average less than 1 fragment will bind to eachbead. Many beads have zero DNA fragments, fewer have a single fragmentand a small number have 2 or more fragments. The steps needed forquantitation often require a separate instrument and separateprocessing.

In one embodiment a target concentration is created by a hybridizationbased pullout. A solid support such as pull-out beads may befunctionalized with a controlled number of binding sites. In someembodiments these are DNA primer complements.

The unamplified sample may have known primers ligated on each end. Insome embodiments the primers may hybridize to the DNA on the pull-outbeads. After the hybridization sites are fully occupied residual DNA maybe washed away, and the DNA bound to the beads may subsequently bedenatured releasing a known quantity of DNA.

In another embodiment the primers ligated to each DNA fragment are boundto the primer complement and detected using fluorescence detection of anintercalating dye. Since the primers are of a known length, the signallevel will be proportional to the number of fragments. In anotherembodiment polymerase and associated dNTPs could be introduced creatingfull length double stranded DNA. When combined with the information fromthe primer signal the full length intercalating dye signal level wouldthen allow determination of the mean fragment length.

In another embodiment dielectrophoresis is used to concentrate DNA.During or after concentration the electrical current is measured todetermine the DNA concentration. In another embodiment the concentratedDNA is quantified by the use of intercalating dyes as described above.In another embodiment, the concentration of the DNA is determineddirectly by optical absorbance. The optical absorbance determinationmay, for example, use an optical source which produces light at 260 nm.

In one embodiment the sample is made very dilute and/or a small volumeof sample reagent and loaded onto beads. DNA would bind to some of thebeads and then be amplified in the virtual reactors creating beads withDNA. The sequencing primer may be made shorter than the complementligated to the sample DNA. Since the sequence is known, the correctdNTPs could be added and detected. In one embodiment multiple dNPTs aresimultaneously added. For example, if all dNTPs are added the polymerasewould extend to the end of the fragment generating a large signal. Saidlarge signal could be generated as a part of the amplification process.This may allow the detection and counting of the number of beads thathave DNA even if the beads had minimal amplification. Knowing how manybeads have signal may allow calculation of the proper dilution togenerate the ideal number of monoclonal beads.

Similarly, measurements made using electrical current, optical signals,or other signals which indicate the concentration of DNA in the samplemay be used to determine the dilution level, if any, needed to optimallyutilize the DNA in the system.

In some embodiments, dilution is needed to properly generate colonies.Similarly dilution may be needed for a nanopore system in order toprevent pore clogging, and conversely, to optimize the duty cyclewhereby a pore may be occupied with a DNA strand. Dilution may beeffectualized as part of an emulsion PCR system, a bridge PCR system, ananopore sequencing system, or a single molecule optical system.

In other embodiments, concentration may be implemented as part ofsystem, and may be effectuated by dielectrophoresis, hybridization,ethanol precipitation or other methods, and may be used to increase theconcentration of DNA to improve an emulsion PCR system, a bridge PCRsystem, a nanopore sequencing system, or a single molecule opticalsystem.

A primary system may determine the concentration of template DNA usingsoftware or hardware to make said determination, and may then eitherconcentrate or dilute as needed prior to utilizing said template DNA inthe next appropriate step said system, which may be amplification orsequencing. Said determination step may also make use of a priorcalibration step, which may use standard comprising DNA of a knownconcentration, or may use DNA of an initially unknown concentration,where the concentration is determined by a separate system. Thedetermined concentration may be entered transferred, or otherwisecommunicated to said primary system. Said primary system may store anyvalues needed for calibration locally in the primary system, or maystore it in a part of a larger system, or in a separate computer, or ina data base. Said calibration information may also include additionalinformation, such as the time of calibration, the operator, the sampleor standard utilized for calibration, or other information as may bedetermined to be relevant.

Many current systems use whole genome amplification in order to havesufficient DNA for their protocol. Typical amplification methods may usedegenerate primers and PCR, random hexamers and isothermal amplificationor other methods for amplification of genomic DNA. Said amplificationmay amplify genomic DNA by a thousand fold or more. This amplificationcan introduce bias and is an additional cost in time and resources. Theability to reduce or eliminate the need to amplify the sample isdesirable. In one embodiment the beads to be loaded are enclosed in apacked bed and sample is pumped across it. The sample can be pumpedthrough the bead bed multiple times to provide additional opportunitiesfor the sample to bind. The high surface area to volume should allow forminimal sample to be used. The beads can subsequently be moved into aflow cell whereby they may be held in place by a magnetic array, andlocal colonies may be created on the beads by PCR or isothermalamplification.

In another embodiment the sample is concentrated in the amplificationregion using the existing electrodes of the emulsion free nano-reactor.In one embodiment electrodes may be established on a single plane. Inanother embodiment electrodes may be added to a second plane parallel tothe plane of the virtual reactors. In other embodiments mixtures of ACand DC voltage inputs are anticipated.

In other embodiments, whole genome amplification or targetedamplification, such as amplification which targets the exome, theconserved regions of the genome, a cancer panel, or other targets ofinterest may be implemented as part of a subsystem within an integratedsystem. Said targeted amplification may also incorporate barcodes fordifferent samples as a part of the amplification process. The amplifiedDNA may then have its concentration determined as explained herein,prior to undergoing clonal amplification for subsequent sequencing,using a clonal sequencing subsystem and method, which may be a part ofthe integrated system. Alternatively or addition, the DNA is sequenceddirectly using a single molecule sequencing subsystem and method whichmay be a part of said integrated system.

Since many projects may not require the full use of a sequencing chip orflow cell it may be desirable to load multiple samples into differentportions or areas of a single chip or flow cell. In one embodiment,samples are directed into separate zones separated by walls on the chipor flow cell using valves integrated into the chip or flow cellassembly. Such valves may be PDMS valves integrated into the fluidicpath. In another embodiment there may be separate zones with separateinputs and outputs. In another embodiment samples may be directed intoseparate zones on a chip or flow cell using a local electric field. Apositive field may be applied to attract DNA to desired regions, while anegative field may be applied to repel DNA from undesired regions. Inanother embodiment samples may be directed into separate zones usingelectromagnets to control the positioning of magnetic or paramagneticbeads. In another embodiment samples may be delivered into individuallanes using self sealing ports. Self sealing ports can include rubbersepta and needles.

In another embodiment samples can be injected at different time pointsand new beads and bead locations can be distinguished using sensorsignals relative to that previously determined for said sensors, wherethe bead locations where previously empty.

In a further embodiment, electrowetting or optoelectrowetting is used todeliver samples to distinct and separate regions of a chip or flow cell.

In some embodiments, containers for the reagents may be cooled asneeded, for example, regents which contain samples, polymerase,phosphatase, or other enzymes may need cooling, for instance, to aboutfour Celsius.

In some embodiments, the amount of reagent contained in the linesleading from the reagent containers to the valve manifold may contain avolume which is significant relative to that which is needed to performa sequencing reaction. In order to prevent needing to discard thisreagent, for example, at the beginning of a new sequencing run, it maybe desirable to cool the lines from where they interface with thereagent containers, up to, or close to, where they enter the valvingmanifold. In some embodiments, where the valving manifold issufficiently separated from where reagents enter the flow cell wheresequencing or another reaction occurs to permit the flow cell and thevalving manifold to operate at different temperatures, for example,about four Celsius and 20 to 40 Celsius respectively, it may bedesirable to cool the valving manifold as well.

In some embodiments it may be desirable to use a system capable of morethan one method of sequencing, wherein one method, process or subsystemfor sequencing provides information of one type, and a second method,process or subsystem sequencing may provide information of a secondtype. For example, in some embodiments it may be desirable to provideone method where the type of information may elucidate the structure ofthe DNA sample, enabling sequence reads which may span the length ofrepeat sequences, such as simple sequence repeats, short tandem repeats,microsatellites, minisatellites, variable number tandem repeats,interspersed repeats such as LINE repeats, SINE repeats such as Alurepeats, direct repeats, or inverted repeats, or other types of repeatsequences which may prevent proper complete assembly of a genome orother desired sequence or sequences of DNA. In some embodiments, it maybe desirable to use a method, process or subsystem for sequencing whichmay, for example, elucidate the structure of DNA, where it may not benecessary to determine the sequence with high accuracy. In someembodiments, it may be desirable to have association of reads which maybe separated many bases apart, as is done in some systems, for exampleby mate pair sequencing or strobe sequencing. In some embodiments it isdesirable to use a method, process or subsystem for sequencing whichprovides sequencing reads which have very high accuracy to detect, forexample, single nucleotide polymorphisms, but for which a longsequencing length of read is unneeded. In some embodiments, it mayfurther be desirable to use a method, process or subsystem forsequencing provides the ability to provide many short reads with lowaccuracy, as may be needed, for example, for whole transcriptomeanalysis.

In some embodiments, it is desirable to use different methods for asingle sample, enabling, for example, detection of both singlenucleotide polymorphisms and structural rearrangements from a singlesample. In some embodiments, it is desirable within a single system toseparate purified nucleic acids into two or more aliquots, which maythen have different corresponding library preparation methods,subsystems, or processes which may include amplification, and mayutilize different amplification methods, subsystems and processes asappropriate for the different desired sequencing methods, subsystems, orprocesses. The size, concentration, or volume of the different aliquotsmay be similar, the same or different, and may be different asappropriate for the different sequencing and/or library preparationmethod(s), subsystem(s), or process(es) which may be utilized to effectthe different desired sequencing results. In some embodiments, themethod may be different for different aliquots, but the subsystems usedmay be the same, and or the subsystem used for the different methods maybe the same subsystem, wherein first one method is used, and then asecond or more subsequent method(s) may be performed used the samesubsystem. For example, it may be desirable to use different fragmentlengths for different sequencing methods, wherein, for example, longfragments may be desirable for determining sequence structure, whereasshort(er) fragments may be desirable for determination of singlenucleotide polymorphisms. Thus it may be desirable to fragment differentaliquots of the sample to different average fragment lengths, whereinthe average fragment length of one aliquot may be longer, potentiallysignificantly longer than another aliquot. In some embodiments, themethod of fragmentation may be the same, and may use for example, asonicator, but the time the sonicator applies energy to said aliquotsand/or the power level of the sonicator applied to said aliquots may bedifferent, such that the level of fragmentation of the sample in the twoor more aliquots may be different, potentially substantially different,and the resultant fragment length may be different, potentiallysignificantly different. In other embodiments, it may be desirable touse a single system to generate long fragments and short fragments; in afurther embodiment, it may be desirable to fragment DNA to sizeappropriate for long fragments, remove an aliquot, and further fragmentremaining DNA to a size appropriate for said short fragments.

In other embodiments, one aliquot of nucleic sample may be genomic DNAfor which single nucleotide polymorphisms may be determined, and asecond aliquot may be RNA for which a transcriptome may be desired.Amplification methods, subsystems, or processes may be different for thegenomic DNA and the RNA, wherein a conversion from RNA to cDNA may beeffected, and wherein the amplification protocol may be different forthe different aliquots, as the accuracy needed for amplification forsingle nucleotide polymorphisms may be much higher than the accuracyneeded for conversion of RNA to cDNA and subsequent amplification ofsaid cDNA. In some embodiments it may be desirable to use lowerconcentrations and/or less expensive reagents for amplification of cDNAfor transcriptome analysis than for amplification of genomic DNA forsingle nucleotide polymorphism analysis. In further embodiments, it maybe desirable to use shorter cycle times for amplification of cDNA fortranscriptome analysis than for amplification of genomic DNA for singlenucleotide polymorphism analysis, which may speed time to answerallowing for transcriptome analysis to commence using the samesequencing subsystem which may be used subsequently by said genomic DNAsingle nucleotide polymorphism analysis. Any other combinations ofseparation of nucleic material for subsequent analysis using differentsequencing methods, subsystems or processes may be envisioned.

In some embodiments, different types of sequencing detection methods,subsystems or processes are used. For example, one subsystem may usesingle molecule sequencing as described by Church et al in U.S. Pat. No.5,795,782, Haneck et al in U.S. Pat. No. 8,137,569 Korlach et al in U.S.Pat. No. 7,361,466, and Clark et al in US2011/0177498, which are eachhereby incorporated by reference in their entirety, which may have lowaccuracy and may have very long sequence reads, while another subsystemmight use optical or electrochemical detection of sequencing bysynthesis as described by McKernan et al in US2009/0181385,Balasubramanian in U.S. Pat. No. 6,833,246, Nyren et al in U.S. Pat. No.6,210,891, Bridgeham et al in U.S. Pat. No. 7,282,370, Williams et al inU.S. Pat. No. 7,645,596, Rothberg et al in U.S. Pat. No. 7,948,015,Toumazou et al in U.S. Pat. No. 8,114,591, and Miyahara et al in U.S.Pat. No. 7,888,013, which are each hereby incorporated by reference intheir entirety. Thus in some embodiments, a single system might have atleast two different detection subsystems, wherein said two differentdetection subsystems may use different sequencing methods, sequencingdetection methods, or sequencing processes, and wherein said differentsequencing methods, sequencing detection methods, or sequencingprocesses may be performed at the same time either for the same sampleor for different samples, or may be performed at different times for thesame sample or different samples.

Exemplary integrated systems are illustrated in accompanying drawings.

FIG. 1A depicts a complete sequencing system 100, which may comprise anexternal computing device 102, and an integrated system 104. Theintegrated system may comprise a rack module 110, which may furthercomprise a fluidics interface subsystem 116, a set of sequencing/sampleprep cards 112, and individual sequencing subsystems 114 on eachsequencing/sample prep card 112. Schematic of sequencing/sample prep 120includes library prep 122, re-useable magnetic arrays 124, which mayfurther comprise sequence detectors 126, which result in sequencing data128.

FIG. 1B depicts a complete library prep subsystem 130, which includessample cell input 131, cellular lysis and protein removal 132 resultingin un-fragmented genomic DNA 133, which may be input to a fragmentationand separation subsystem 134, which may then output fragmented genomicDNA 136, which may be transported along with a set of beads 135, to avirtual well array 137 for amplification, and then said beads may beseparated utilizing a field 138 in an bead enrichment module 139 intosets of beads with amplification from set of beads withoutamplification.

FIG. 1C schematically illustrates a genomic DNA fragmentation andseparation system 140, comprising input un-fragmented genomic DNA andfragmentation beads 142, which are input to a fragmentation subsystem144 wherein said un-fragmented genomic DNA may be fragmented. Thefragmented DNA may be separated by size in a channel 146 using pumpingor electrophoretic force from pumps or electrodes 147, and may then bemoved to an output from said separation channel 146 via fluidic outputs148, and outputting said fragmented DNA 149.

FIG. 1D shows an embodiment of a PDMS library preparation module 150which includes a lysis section 152, a protein removal section 154, andan amplification section 156.

While various embodiments have been described above, it should beunderstood that they have been presented by way of example only, and notlimitation. Where methods and/or schematics described indicate certainevents, and/or flow patterns, and/or chemical reactions occur in acertain order, the ordering of certain events and/or flow patternsand/or chemical reactions may be modified. While the embodiments havebeen particularly shown and described, it will be understood thatvarious changes in form and or detail may be made.

Although various embodiments have been described as having particularfeatures and/or combinations of components, other embodiments may bepossible having a combination of any features and/or components asdiscussed above.

1.-36. (canceled)
 37. A method for sequencing a nucleic acid sample,comprising: a. providing a plurality of particles adjacent to a sensorarray, wherein an individual particle of said plurality is positionedadjacent to an individual sensor of said sensor array and is coupled toa nucleic acid molecule generated from said nucleic acid sample, andwherein said individual sensor provides a virtual wall during sequencingthat isolates said individual sensor from other sensors of said sensorarray; b. hybridizing a primer to said nucleic acid molecule; c.performing a primer extension reaction by contacting said nucleic acidmolecule with nucleotide bases in the presence of a polymerase adjacentto said nucleic acid molecule; d. detecting signals indicative ofincorporation events associated with one or more of said nucleotidebases during said primer extension reaction; e. monitoring andcorrecting for phase error introduced during said primer extensionreaction; and f. generating a sequence of said nucleic acid sample atreduced phase error.
 38. The method of claim 37, wherein said signalsare local impedance changes accompanying said primer extension reaction.39. The method of claim 37, wherein said individual sensor measures alocal impedance change within a Debye layer associated with saidindividual particle.
 40. The method of claim 37, wherein said individualsensor comprises at least two electrodes that are associated with aDebye layer of said individual particle.
 41. The method of claim 37,wherein said virtual wall is generated by a local electric field orlocal magnetic field.
 42. The method of claim 37, wherein said virtualwall isolates or concentrates components of said primer extensionreaction.
 43. The method of claim 37, wherein phase error is correctedby (i) adding a combination of three nucleotide bases, (ii) reversiblyincorporating into an in-phase polynucleotide strand a chain terminatingnucleotide base, or (iii) adding an oligonucleotide clamp thathybridizes to said nucleic acid molecule and halts said primer extensionreaction.
 44. The method of claim 43, further comprising denaturing,destabilizing, or degrading said clamp to continue said primer extensionreaction.
 45. The method of claim 44, wherein said clamp has a 3′terminating nucleotide base that cannot be extended, and wherein said 3′terminating nucleotide base can be optionally removed, thereby becominga primer for a subsequent downstream primer extension reaction.
 46. Themethod of claim 37, wherein said phase error is corrected by selectingone or more nucleotide bases for incorporation to re-phase a lag by oneor two bases.
 47. The method of claim 37, further comprising monitoringsaid signals for loss of signal that is indicative of phase error, andcorrecting by re-phasing to restore said signals.
 48. The method ofclaim 37, further comprising stockpiling said polymerase on or near saidnucleic acid molecule.
 49. The method of claim 48, further comprisingbinding a repair protein or single stranded binding protein to saidnucleic acid molecule.
 50. The method of claim 37, wherein said nucleicacid molecule is circularized.
 51. The method of claim 50, wherein saidpolymerase is a strand displacing polymerase.
 52. The method of claim37, further comprising anticipating phase error based on a referencesequence.
 53. A method for sequencing a nucleic acid sample at reducedphase error, comprising: a. providing a plurality of particles adjacentto a sensor array, wherein an individual particle of said plurality ispositioned adjacent to an individual sensor of said sensor array and iscoupled to a nucleic acid molecule generated from said nucleic acidsample; b. hybridizing a primer to said nucleic acid molecule; c.performing a primer extension reaction by contacting said nucleic acidmolecule with nucleotide bases in the presence of a polymerase adjacentto said nucleic acid molecule; d. using said individual sensor,measuring local impedance changes within a Debye layer associated withsaid individual particle during said primer extension reaction; e.monitoring and correcting for phase error introduced during said primerextension reaction; and f. generating a sequence of said nucleic acidsample at reduced phase error.
 54. The method of claim 53, wherein saidindividual sensor comprises at least two electrodes that are coupled toa Debye layer of (i) said individual particle or (ii) nucleic acidmolecules coupled to said individual particle.
 55. The method of claim53, wherein said individual sensor provides a virtual wall duringsequencing that isolates said individual sensor from other sensors ofsaid sensor array.
 56. The method of claim 53, further comprisingmonitoring signals associated with said local impedance changes for lossof signal that is indicative of phase error, and correcting said phaseerror by re-phasing to restore said signals.
 57. A sensing system,comprising: an array of sensors, wherein an individual sensor of saidarray comprises at least two electrodes that, during sensing using saidindividual sensor, are disposed adjacent to a particle comprising anucleic acid molecule coupled thereto, wherein during sensing said atleast two electrodes are coupled to a Debye layer associated with saidparticle, wherein said individual sensor is configured to generate avirtual wall to control the movement of said particle or said nucleicacid molecule(s) during sensing, and wherein said individual sensor isconfigured to control a shape or period of said virtual wall.
 58. Thesystem of claim 57, wherein said particle has a low zeta potential. 59.The system of claim 57, wherein, during sensing, said electrodes areconfigured to measure an impedance change associated with said particle.60. The system of claim 57, wherein said individual sensor is configuredto generate said virtual wall by an alternating current field.
 61. Thesystem of claim 57, wherein said individual sensor is configured toapply a magnetic field to immobilize said particle.
 62. The system ofclaim 57, wherein said individual sensor is configured to control saidshape or period in response to a change from a sensor among said arrayof sensors.
 63. The system of claim 57, wherein said individual sensorcomprises a well.
 64. The system of claim 63, wherein said well has adiameter that is less than a diameter of said particle.
 65. The systemof claim 57, further comprising a polymer adjacent to said individualsensor, wherein said polymer reduces the migration rate of nucleotidesconfined proximate to said individual sensor during sensing.
 66. Thesystem of claim 57, wherein said at least two electrodes comprise afirst electrode and a second electrode that are separated by adielectric layer.
 67. The system of claim 66, wherein said at least twoelectrodes further comprise a third electrode that is for performingdielectrophoretic concentration or confinement during sensing.
 68. Amethod for sequencing a nucleic acid sample, comprising: a. providing aplurality of particles adjacent to a sensor array, wherein an individualparticle of said plurality is positioned adjacent to an individualsensor of said sensor array and is coupled to a nucleic acid moleculegenerated from said nucleic acid sample; b. hybridizing a primer to saidnucleic acid molecule; c. performing a primer extension reaction bycontacting said nucleic acid molecule with nucleotide bases in thepresence of a polymerase adjacent to said nucleic acid molecule; and d.sequencing said nucleic acid sample by detecting signals indicative ofincorporation events associated with one or more of said nucleotidebases during said primer extension reaction, which sequencing employssteady state electronic sequencing.
 69. The method of claim 68, furthercomprising monitoring and correcting for phase error introduced duringsaid primer extension reaction.